Preservative Removal from Eye Drops

ABSTRACT

A BAK removal device is constructed as a plug of microparticles of a hydrophilic polymeric gel that displays a hydraulic permeability greater than 0.01 Da. The polymer hydrophilic polymeric gel comprises poly(2-hydroxyethyl methacrylate) (pHEMA). The particles are 2 to 100 μm and the plug has a surface area of 30 mm 2  to 2 mm 2  and a length of 2 mm to 25 mm and wherein the microparticles of a hydrophilic polymeric gel has a pore radius of 3 to 60 μm.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/429,384, filed Dec. 2, 2016, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand drawings.

BACKGROUND OF INVENTION

Ophthalmic diseases are most commonly treated by instillation of eyedrops with frequencies varying from one or two a day for diseases likeglaucoma to as many as ten a day for severe infections. The drugsolutions in eye drop bottles can get contaminated during use due tocontact of the tip with hands or tears while instilling the drops. In arecent study with 204 glaucoma patients, only 39% were able to instillthe eye drops without touching the bottle to the eye surface. There areadditional risks of cross-contamination when multiple patients share abottle, such as in a family or in hospitals. The high potential for thecontamination after opening the bottles has led to regulations thatrequire the addition of an antimicrobial agent in multi-dose eye dropformulations. Several preservatives have been researched and used incommercial formulations, including: alcohols, parabens, EDTA,chlorhexidine, and quaternary ammonium compounds. In addition toantimicrobial efficacy, the preservatives require suitable physicalproperties for incorporation into the formulations, such as chemical andthermal stability, compatibility with the eye drop container and othercompounds in the formulation, and, more importantly, negligible toxicityto ocular tissues.

Regulations require that ophthalmic preservatives achieve 1.0 and 3.0log reduction by days 7 and 14, respectively, along with no increase insurvivors from days 14-28 and no increase in survivors for the fungifrom day 0 to day 28 after inoculation with 10⁶ colony forming units(cfu)/mL. (Baudouin et al. “Preservatives in Eyedrops: the Good, the Badand the Ugly”. Progress in Retinal and Eye Research, 2010, 29, 312-34)Due to high efficacy and low corneal toxicity, the quaternary ammoniumcompounds are preferred preservatives.

Benzalkonium chloride:

where a mixture of n being 8, 10, 12, 14, 16, and 18, is the most commonchoice with n=12 and 14 being the primary homologues. Eye dropformulations require BAK at concentrations ranging from 0.004 to 0.025%(w/w) to achieve the regulatory effectiveness. In spite of the positivesafety profile of BAK, achievement of the targeted antimicrobial andantifungal effects is not possible without levels that cause some toxicside effects to the cornea. BAK can cause tear film instability, loss ofgoblet cells, conjunctival squamous metaplasia and apoptosis, disruptionof the corneal epithelium barrier, and damage to deeper ocular tissues.

The potential for ocular damage from the preservatives is particularlyhigh among patients suffering from chronic diseases that require dailyeye drop instillations for periods of years to decades, such as glaucomapatients. Several clinical and experimental studies have shown thattoxic side effects from preservative free eye drops are significantlylower than from their preserved counterparts. A multicentercross-sectional epidemiologic study using preservative orpreservative-free beta-blocking eye drops showed that patients onpreservative free eye drops exhibit significantly fewer ocular symptomsand signs of irritation compared to those using preserved eye drops.(Jaenen et al. “Ocular Symptoms and Signs with Preserved andPreservative-free Glaucoma Medications”, European Journal ofOphthalmology. 2007, 17, 341-9) Preserved glaucoma drug timolol causessignificantly higher tear film instability and disruption of cornealbarrier function than preservative-free timolol in healthy subjects.(Ishibashi et al., “Comparison of the Short-term Effects on the HumanCorneal Surface of Topical Timolol Maleate with and without BenzalkoniumChloride”, Journal of Glaucoma, 2003, 12, 486-90) Similar results werefound when comparing preservative-free and BAK-containing carteolol.(Baudouin et al., “Short Term Comparative Study of Topical 2% Carteololwith and without Benzalkonium Chloride in Healthy Volunteers”, BritishJournal of Ophthalmology. 1998, 82, 39-42) Goblet cell loss andincreased cytoplasmic/nucleus ratio, two characteristics of dry eyedisease, have been shown to occur when using BAK containing tearsubstitutes. (Rolando et al., “The Effect of Different BenzalkoniumChloride Concentrations on Human Normal Ocular Surface”. The LacrimalSystem, Kugler and Ghedini, New York 1991, 87-91) A significantreduction in Schirmer test values was observed for subjects receivingBAK eye drops compared with subjects not receiving therapy. (Nuzzi etal., “Conjunctiva and Subconjunctival Tissue in Primary Open-angleGlaucoma after Long-term Topical Treatment: an Immunohistochemical andUltrastructural Study”, Graefe's Archive for Clinical and ExperimentalOphthalmology, 1995, 233, 154-62) Patients using preserved eye drops andexperiencing toxicity symptoms, such as allergy, blepharitis or dry eye,experienced rapid improvements upon switching to preservative-freeformulations. Such studies suggest a role of preservatives in thepreponderance of dry eye symptoms in glaucoma patients, who typicallyuse multiple drugs with multiple instillations each day.

BAK is considered a ‘necessary evil’ for prevention of microorganismgrowth in the bottles while displaying toxic effects on the oculartissue. The industry has taken a few approaches to solve this problem.One approach is to develop more efficacious glaucoma therapies, such as:use of prostaglandins that require instillation of only one eye dropeach day; and combinations that contain multiple drugs in the sameformulation to eliminate instillation of multiple eye drops.Nevertheless, both of these approaches still permit a cumulative effectto preservatives over long periods of time. Furthermore, only a fewcombination products are available, generally combinations from a singlemanufacturer.

A second approach is to provide single dose packages, and severalglaucoma formulations are now available as preservative free singledoses. While this approach can eliminate exposure to preservatives, inaddition to increasing manufacturing costs and the environmental impactof packaging, single dose formulations contain about 0.3 to 0.4 mL offormula, which is significantly more than the typical eye drop volume of30 Lμ, leading to wastage or possibly misuse of the same package formultiple days. This approach can suffer if bacterial contaminationoccurs prior to packaging.

Another approach is to replace BAK with a less toxic preservative, suchas: Purite®, a stabilized oxychloro complex; and Sofzia®, which iscomposed of boric acid, propylene glycol, sorbitol, zinc chloride andpolyquaternium compounds, some of which are used in contact lens caresolutions. While these alternatives may be promising, no data on longterm impact from use of these preservatives is available, and consistentuse of these preservatives over extended periods of years may well provethem toxic.

The solution in a bottle is typically contaminated during theinstillation of the eye drops due to the contact of the bottle tip withthe eye surface, contact of the tip with hands, or both. As the eye dropdetaches from the bottle, a small volume of liquid remaining at the tipis sucked back, which can take the bacteria into the bottle, leading tothe contamination. An ABAK® (Laboratoires Thea, France) designintroduces a 0.2 μm filter at the top of the bottle to filter outbacteria from the re-entering solution, thereby preventingcontamination. Though effective, this approach does not protect againstcontamination prior to packaging. Also the 0.2 μm filter could requireadditional pressure to push the drops, making drop instillationdifficult, particularly for the elderly. Additionally, any leak in thefilter or bacteria transport through the pores could allow theformulation in the bottle to get contaminated. It is also not clearwhether this design can protect against growth of bacteria trapped inthe filter. The COMOD® (Ursapharm, Germany) system combines an air freepump and an inner lining that retracts as the liquid is pushed out toavoid contamination of the contents of the bottle. While this design isinnovative and useful, its complexity and increased cost are majorconcerns. As with ABAK®, COMOD® cannot protect against anymicroorganisms introduced due to errors in the manufacturing processescausing loss of sterility. This makes the filling of these devicescomplicated because sterility is essential at each step.

U.S. Pat. No. 5,080,800 teaches a process for removing components fromsolutions, including preservatives from eye-drops. The process involvesthe use of ion exchange resins to selectively remove ocularpreservatives. Ion exchange resins have not been tested extensively forbiocompatibility and cytotoxicity and inherently are non-selective,adsorb ionic drugs as readily as any ionic preservative such as BAK. Thehydraulic permeability of these resins is not addressed although thischaracteristic is critical for devices that allow formation of dropswithout excessive pressure. U.S. Pat. No. 5,080,800 also does not teachon the importance of ensuring that the filters are designed to resistgrowth of microorganisms that may remain trapped. U.S. Pat. No.5,080,800 does not teach on the possibility of dilution of the BAKconcentration in the formulation because of draining of the BAK freeformulation from the filter into bottle after each eye dropinstillation. Hence a practical way of retaining the beneficial behaviorof preservatives while avoiding their toxic effects in the eye remains aneed.

BRIEF SUMMARY

Embodiments of the invention are directed to a preservative removingdevice having a plug of microparticles that are a hydrophilic polymericgel. The plug has a shape that matches an outlet to a container for asolution, emulsion, or suspension. The hydrophilic polymeric gel swellsin the presence of the solution, emulsion, or suspension and selectivelyabsorbs a preservative contained therein. The plug of microparticles hasa hydraulic permeability greater than 0.01 Da, even greater than 10 Dain some embodiments. The hydrophilic polymeric gel can be poly hydroxylethyl methacrylate (pHEMA) or a pHEMA copolymer such as poly hydroxylethyl methacrylate-co-methacrylic acid, or other biocompatible polymer,including but not limited to, dimethyl acrylamide, methyl methacrylate,and silicones. The hydrophilic polymeric gel has interconnected pores,wherein the pores have an average radius of 1 to 60 μm. Themicroparticles can be from 2 to 100 μm in cross-section. Thepreservative removing device can remove the preservative benzalkoniumchloride (BAK).

In an embodiment of the invention, the hydrophilic polymeric gel is apreservative containing device, for example, a gel that is preloadedwith the BAK at a concentration of one to 100 times that of thesolution, emulsion, or suspension in the container. The preservativeincorporation into the device would impart sterility, which is arequirement for all ophthalmic preparations and dispensers. Thepreservative incorporated device could also act as a preservativeremoval device if the initial loading is below the equilibrium capacity.Additionally, the plug can include antibacterial microparticles, suchas, silver particles.

In an embodiment of the invention, the polymeric material can bepretreated with a drug in the solution, emulsion, or suspension in thecontainer, wherein the polymer is less than saturated or saturated withthe drug to reduce or eliminate further drug uptake during thedispensing of the solution, emulsion, or suspension.

In an embodiment of the invention the preservative removing device isincluded in a multi-dosing device for delivery of an ophthalmic solutionis a compressible bottle that has an outlet extension containing thepreservative removing device. When the hydrophilic polymeric gel is dry,it has dimensions smaller than the internal dimensions of the outletextension but has larger than the internal dimensions of the outletextension when swollen with the ophthalmic solution. The multi-dosingdevice can include an ophthalmic agent selected from timolol,dorzolamide, dexamethasone phosphate, dexamethasone, latanoprost orother prostaglandins, rewetting eye drops, or any other compounds thatis delivered to the eye for disease treatment or comfort improvement.

In another embodiment of the invention, a method of administering anophthalmic agent involves providing a compressible bottle with apreservative removing device at the outlet of the compressible bottlecontaining an ophthalmic agent and a preservative, which upon applyingpressure to the compressible bottle; the solution is forced through thepreservative removing device.

In another embodiment of the invention, a method of administering anophthalmic agent involves providing a compressible bottle with apreservative removing device at the outlet of the compressible bottlecontaining an ophthalmic agent and a preservative, and a preservativeloaded film at the bottom of the bottle which upon applying pressure tothe compressible bottle; the solution is forced through the preservativeremoving device.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows a photograph of a prototype design with a filter,according to an embodiment of the invention, incorporated into the neckof the eye drop bottle and FIG. 1B shows a CAD design of a bottle,filter, tip and cap assembly of the device.

FIG. 2A shows a photograph of a prototype design with the filterincorporated into the tip of the eye drop bottle and FIG. 2B shows a CADdesign of a bottle, filter, tip, and cap assembly of the device

FIG. 3 shows a plot of hydraulic permeability of the BAK filter plug ifthe plug area is 78.5 mm2 and model predicted design parameters oflength, pore radius where the solid lines indicate the upper limit andthe minimum requirement of the pore size and the respective hydraulicpermeability.

FIG. 4 shows an SEM image of macroporous pHEMA hydrogel.

FIG. 5 shows a schematic of the experiment setup for measuring thehydraulic permeability of any material by packing it in a syringe. Thesyringe is filled with water and then a known force is applied to pushout the water.

FIG. 6 shows a plot of measured hydraulic permeability for macroporouspHEMA hydrogel packed in a syringe. For each packed sample, thepermeability was measured 10 times to determine whether compactionoccurs due to flow. The data was measured for 12 independent sampleswith data points representing the mean±SD for n=12 data points persample.

FIG. 7 shows a bar chart of the percentages of BAK and timolol that areremoved after passing 2.5 mL of timolol/BAK solution through 5 mm thickmacroporous pHEMA gel packed into a 1 cm diameter syringe for a seriesof 10 consecutive passes, where the data are presented as mean±SD withn=3.

FIG. 8 shows a bar chart of the percentages of BAK and dorzolamide thatare removed after passing 2.5 mL of dorzolamide/BAK solution through 5mm thick macroporous pHEMA gel packed into a 1 cm diameter syringe for aseries of 10 consecutive passes, where the data are presented as mean±SDwith n=3.

FIG. 9 shows a bar chart of the percentages of BAK and latanoprost thatare removed after passing 2.5 mL of latanoprost/BAK solution through a 5mm thick macroporous pHEMA gel packed into a 1 cm diameter syringe for aseries of 10 consecutive passes, where the data are presented as mean±SDwith n=3.

FIG. 10 shows a bar chart of the percentages of BAK and dexamethasonethat are removed after passing 2.5 mL of dexamethasone/BAK solutionthrough a 5 mm thick macroporous pHEMA gel packed in a 1 cm diametersyringe for a series of 10 consecutive passes, where the data arepresented as mean±SD with n=3.

FIG. 11 shows a bar chart of the percentages of BAK and timolol that areremoved after passing 2.5 mL of timolol/BAK solution through 5 mm thickplug formed by packing crushed macroporous pHEMA gel in a 1 cm diametersyringe for a series of 10 consecutive passes where each pass isseparated by 24 hours, where the data are presented as mean±SD with n=3.

FIG. 12 shows a bar chart of the percentages of BAK and dorzolamide thatare removed after passing 2.5 mL of dorzolamide/BAK mixture solutionthrough 5 mm thick plug formed by packing crushed macroporous pHEMA gelin a 1 cm diameter syringe for a series of 10 consecutive passes whereeach pass is separated by 24 hours, where the data are presented asmean±SD with n=3.

FIG. 13 shows a bar chart of the percentages of BAK and latanoprost thatare removed after passing 2.5 mL of latanoprost/BAK solution through 5mm thick plug formed by packing crushed macroporous pHEMA gel in a 1 cmdiameter syringe for a series of 10 consecutive passes where each passis separated by 24 hours, where the data are presented as mean±SD withn=3.

FIG. 14 shows a bar chart of the percentages of BAK and dexamethasonethat are removed after passing 2.5 mL of dexamethasone/BAK solutionthrough 5 mm thick plug formed by packing crushed macroporous pHEMA gelin a 1 cm diameter syringe for a series of 10 consecutive passes whereeach pass is separated by 24 hours, where the data are presented asmean±SD with n=3.

FIG. 15 shows a plot of the hydraulic permeability of crushedmacroporous pHEMA particles packed in a syringe with measurements for aseries of 10 consecutive passes where each pass is separated by 24hours, where the data are presented as mean±SD, with n=12.

FIG. 16 shows a bar chart of the percentages of BAK and dexamethasonethat are removed after pushing 2.5 mL of dexamethasone/BAK mixturesolution through a 5 mm thick macroporous HEMA-co-MAA copolymer hydrogelpacked in a 1 cm diameter syringe, where measurement was repeated 3times in immediate succession.

FIG. 17 shows a bar chart of the percentages of BAK and dexamethasonethat are removed after pushing 2.5 ml of dexamethasone/BAK mixturesolution through the 5 mm thick macroporous pHEMA hydrogel treated with1% of MAA solution packed in a 1 cm diameter syringe, where measurementwas repeated 3 times in immediate succession.

FIG. 18 shows an SEM photographic image of pHEMA particles synthesizedby thermally initiated polymerization with EGDMA as cross-linker.

FIG. 19 shows an eye drop bottle prototype packed with BAK removal plugon the tip. The extra space after the plug was kept in this design tofacilitate measurement of the hydraulic permeability.

FIG. 20 shows a plot of the total flowrate from the bottle containingthe plug as a function of time. The hydraulic permeability wascalculated by fitting the data to the theoretical equation.

FIG. 21 shows a bar chart of the percentages of BAK and latanoprost thatare removed after passing 1.5 mL of a latanoprost/BAK solution through8-mm thick crushed macroporous pHEMA particles packed in the tip of theeye drop prototype for 10 daily runs over 10 days where the data pointsare mean±SD with n=3.

FIG. 22 shows a SEM image of pHEMA particles synthesized by UVpolymerization using EGDMA as cross-linker, where the pHEMA particlesize ranges from 10 to 200 μm with near spherical particles with smoothsurface.

FIG. 23 is a bar chart of the percentages of BAK and timolol that areremoved after passing the timolol/BAK solution through 8-mm thick plugof pHEMA particles prepared by photo-polymerization packed in the tip ofthe eye drop prototype bottle with 1.5 mL of drug/BAK solution passingthrough the plug for each of 5 passes in immediate succession.

FIG. 24 is a bar chart plot of the percentages of BAK and timolol thatare removed after passing a timolol/BAK solution through 8-mm thick plugof pHEMA particles prepared by heat-initiated polymerization packed inthe tip of an eye drop prototype bottle, where 1.5 mL of drug/BAKsolution was passed through the packing in each run for 10 passes inimmediate succession.

FIG. 25 shows the SEM image of pHEMA particles prepared by UVpolymerization using SR454HP as cross-linker.

FIG. 26 shows a bar chart of the percentages of BAK and timolol that areremoved after passing 1.5 mL of a timolol/BAK mixture solution through1.8 cm thick plug of pHEMA particles prepared by using SR454HP ascross-linker packed in the tip of the eye drop prototype for 10 dailyruns over 10 days where the data points are mean±SD with n=3.

FIG. 27 shows a bar chart of the percentages of BAK and timolol that areremoved after passing 1.5 mL of a timolol/BAK mixture solution through1.8 cm thick plug of pHEMA particles prepared by using SR9035 ascross-linker packed in the tip of the eye drop prototype for 10 dailyruns over 10 days where the data points are mean±SD with n=3.

FIG. 28 is a plot of the partition coefficient of Bimatoprost in variouscopolymer compositions for particulate gels of HEMA and MAA.

FIG. 29 is a plot of the partition coefficient of BAK in variouscopolymer compositions for particulate gels of HEMA and MAA.

FIG. 30 is a plot of the percent uptake of Bimatoprost in particulategels of HEMA and MAA from drops passed through the particles packed in adropper tip.

FIG. 31 is a plot of the percent uptake of Bimatoprost in particulategels of HEMA and MAA from drops passed through the particles packed in adropper tip.

FIG. 32 shows a plot of the percent uptake of Bimatoprost in particulategels of 25/75 pMAA/tBM from drops passed through the particles packed ina dropper tip.

FIG. 33 is a plot of the equilibrium interfacial surface tension of BAKsolutions that fits a Langmuir surfactant adsorption isotherm model forestimation of BAK concentrations.

FIG. 34 shows a plot of the equilibrium interfacial surface tension datafor commercial Bimatoprost/BAK solutions from Allegran over the periodof a week.

FIG. 35 shows a bar chart of the calculated BAK removal from equilibriuminterfacial surface tension data for commercial Bimatoprost/BAKsolutions from Allegran over the period of a week.

FIG. 36 shows a plot of the percent uptake of Timolol from drops forun-equilibrated HEMA particles.

FIG. 37 shows a plot of the percent uptake of Timolol from drops fortwo-week equilibrated HEMA particles.

FIG. 38 shows a plot of the percent uptake of Timolol from drops forfive-day equilibrated HEMA particles.

FIG. 39 shows a plot of the percent uptake of Visine from drops forun-equilibrated HEMA particles.

FIG. 40 shows a plot of the percent uptake of Visine from drops forone-week equilibrated HEMA particles.

FIG. 41 shows a plot of the percent uptake of Visine A from drops forun-equilibrated HEMA particles.

FIG. 42 shows composite UV spectra of Visine A from drops for one-monthequilibrated HEMA particles.

FIG. 43 shows a plot of the percent uptake of bimatoprost from variouscompositions of un-equilibrated HEMA/MMA particles.

FIG. 44 shows a plot of the percent uptake of bimatoprost from five-dayequilibrated 90/10 HEMA/MAA particles.

FIG. 45 shows a plot of the percent uptake of bimatoprost from five-dayequilibrated HEMA particles.

DETAILED DISCLOSURE

Embodiments of the invention are directed to a multi-dosing device andmethod that eliminates patients' exposure to preservatives, particularlyBAK, in delivered eye drops while retaining BAK in the containedformulation and ensuring that the eye drop bottle remains sterile.Benefit of the BAK for storage is retained while the potential forocular toxicity from BAK is eliminated. In an embodiment of theinvention, a porous preservative removing device, also referred toherein as a plug, is situated in the neck of the eye drop bottle leadingto the drop exit, as shown in FIG. 1. In another embodiment of theinvention, the plug is situated in a section of the tip of the eye dropbottle, as shown in FIG. 2. A large tip is included in the bottle toallow a long plug to be positioned therein The preservative removingdevice can be separate filter that is attached to the formulationdispensing unit through a suitable connector for use. The plug mustdisplay a high hydraulic permeability such that relatively littlepressure is required to dispense a fluid. The needed hydraulicpermeability depends on the design of the filter, where larger poresallow higher liquid flow for a given pressure drop. In embodiments ofthe invention, hydraulic permeability is larger than about 0.01 Da and apermeability of about 0.1 Da is adequate for the typical embodiment ofthe invention where the plug is one that fits a size that fits state ofthe art eye drop packages. A hydraulic permeability of 1 to 10 Da canensure that the fluid that remains in the filter after instillation ofthe eye drop is sucked back into the bottle. A larger hydraulicpermeability allows the same plug to work for a wide range offormulations including high viscosity formulations, such as, rewettingeye drops.

The plug is of a material with high affinity for the preservative BAKand low affinity for the drug or other ophthalmological agent, such thatat least 50 percent of the preservative is removed from the solution bythe plug and at least 50 percent of the drug is retained by the solutionthat is dispensed from the device. The high affinity is a necessary butnot a sufficient requirement because the concentration in the elutingliquid may not be in equilibrium with that in the plug due to the shortcontact time of 1-3 sec. In addition to the high partition coefficient,the adsorption rate constant must be sufficiently high so that the timefor adsorption of a drug molecule to the polymer is less than thecontact time of 1-3 sec. Furthermore it is also important that the poresize in the plug is small enough so that the molecules that areinitially far away from the surface of the polymer in the plug candiffuse towards the polymer and adsorb. When the plug material has ahigh partition coefficient and adsorption rate and the pore size in theplug is optimized, all or most of the preservative will adsorbs on thepore surfaces in the plug and the eluting drops will bepreservative-free. The preservative free liquid that elutes through theplug is instilled directly into the eyes. The highly porous plugmaterial selectively extracts the preservative, allowing the eye dropformulation to flow through the plug with only a small pressure drop,yet allowing sufficient time and surface area to bind the preservative.

The material selected is critical, allowing for construction of a safe,biocompatible filter for preservative removal. Previous patents haveproposed ion exchange resins for similar applications but such materialsmay also remove ionic drugs. For example, BAK is cationic and a numberof ophthalmic drugs such as timolol are cationic at physiological pH andthus the ion exchange resins may remove both. A number of materials havebeen widely used for ophthalmic applications and such materials arecompatible with the eye. Poly(2-hydroxyethyl methacrylate) (pHEMA) isone of the most commonly used material for devices used in the eye, buthas never been explored for its use as a permeable liquid plug forremoval of any ionic materials. Since pHEMA in non-ionic, high bindingof BAK or other ionic compounds is not possible in the manner of an ionexchange material. We started with pHEMA due to its excellentbiocompatibility and assumed that we would need to incorporate othercomponents into the material to obtain the desired selectivity for BAK.Surprisingly, it was observed that pHEMA is extremely effective inadsorbing BAK without any modifications. The pHEMA material has a highpartition coefficient for BAK and the adsorption times were determinedto be less than the transit time of 3 s, implying that BAK solutionflowing through a pHEMA plug will have sufficient time to adsorb on thepolymer. Furthermore, pHEMA is already used as an ophthalmic material,making it the ideal choice for the plug material.

In an embodiment of the invention, the plug material is a hydrogel, suchas poly(2-hydroxyethyl methacrylate) (pHEMA). The pHEMA hydrogeldisplays an extremely high binding capacity for BAK with a partitioncoefficient of about 100-500 depending on the BAK concentration and thestructure of the pHEMA matrix used in the measurement. In contrast, thepartition coefficients of most hydrophilic ophthalmic drugs into thepHEMA matrix range from about 1 to 10, and partition coefficients ofhydrophobic drugs are in the range of 10 to 50. When a drug's partitioncoefficient into the plug is lower by at least an order of magnitudethan the plugs affinity for BAK, the porous pHEMA plug permits selectiveremoval of BAK from eye drop formulations.

In an embodiment of the invention, the pHEMA plug is highly porous,having large interconnected pores that allow easy solution flow with thepreservative BAK adsorbing on the walls of the pores. The plug can beformed as a porous gel, a packed bed, or a structure formed by 3Dprinting, soft lithography, electrospinning, or any other method. Use ofa macroporous gel, according to an embodiment of the invention, permitsa relatively simple scalable preparation process that is cost effective.Macroporous gels are biphasic materials consisting of largeinterconnected pores dispersed throughout the polymer. The macroporoushydrogels can be prepared by free radical polymerization of a monomer ina diluent that dissolves the monomer but not the polymer. If theconcentration of the diluent is more than the equilibrium swellingcapacity of the polymer, the extra diluent phase separates and formspores. Although macroporous pHEMA hydrogels can be prepared using wateras the diluent, such gels are typically weak mechanically. Organicdiluents with good solubility for HEMA but poor solubility for pHEMAinclude dodecan-1-ol and 1,2-dichloroethane, and such solvents result inrobust gels. However, significant amount of organic liquids isundesirable for biomedical applications. Therefore, the macroporoushydrogels are prepared by enhanced phase separation using aqueous NaClsolution. In another embodiment of the invention, the macroporous gelcould be prepared from other suitable polymers such as poly acrylamideand pHEMA particles could be dispersed as the matrix for sequestrationof the preservative.

Alternatively, in an embodiment of the invention, the plug can beprepared as a packed bed of pHEMA or other polymeric particles. Theparticles can be macroporous. The packed beds of macroporous particlescan have three levels of porosity: the space between the sphericalparticles providing inter-connected channels for the liquid flow; themacropores in the spherical particles to allow BAK diffusion into theparticles and adsorb on the surface of these pores; and the pHEMApolymer's inherent porosity having nano-sized pores which provide thesurface area for high BAK uptake into the gel. In a packed bed, themultiple levels of porosity avoids any tradeoff between increasedpermeability and reduced area, and, thus, increasing the particle sizeto increase the hydraulic permeability with minimal impact on thesurface area for adsorption of BAK. Non spherical particles could bevery useful as well in achieving high porosity that will increase thehydraulic permeability.

Nano or micron sized polymeric particles (nanogels or microgels) areproduced by solution or bulk polymerization, where bulk gelation isavoided by using dilute monomer solutions or by using chain transferagents and restricting the conversion of monomer to polymer. Forexample, the water fraction is significantly high to prevent macroscopicgelation of the microgels. By varying the water fraction, and otherformulation parameters, particles ranging from 5 to 50 μm in size can beproduced. We observed that the type of the cross-linker has a verysignificant impact on the type and size of particles produced.Additionally or alternatively, a chain transfer agent can be used toeffectively cap the growing chains on the surfaces of microparticles. Bymanipulating the degree of dilution, salt concentration, and theconcentration of chain transfer agent, a wide particle size range can beproduced. The particles will be dried and then packed in a bed to createthe monolith for BAK separation.

In another embodiment of the invention, cryogels are prepared byfreezing a polymerization mixture and using a redox couple as theinitiator to polymerize under frozen conditions. Cryogels typically havelarge pores in the range of tens to hundreds of microns. The initiatorcan be a mixture of N,N,N′,N′-tetramethylethylene diamine (TEMED) andammonium persulfate (APS). The mixture is frozen at −15° C. for 12 hoursand then thawed.

In embodiments of the invention, various filters may be placed tosupport the porous matrix or the particles. The filter is designed tooffer minimum resistance to fluid flow.

Other embodiments of the invention are directed to a method ofincorporating the preservatives into particles that are added to theformulation in the containers such that the particle-incorporatedpreservative can provide the required preservative effect, but not flowout with the formulation. The particles can directly impart thepreservative effect such as colloidal silver particles. The particles inthe formulation are prevented from eluting, either by attachment to thecontainer walls through long polymeric chains, or by placing a filter atthe exit from the device of size smaller than the particles. In anotherembodiment of the invention, the walls of the container or othersurfaces can have attached or incorporated preservative, to provide thepreservative effect to the formulation. For example, the preservativesource can be a pHEMA membrane with 1-10% by volume of the initialformulation volume, equilibrated with BAK at the starting concentrationin the formulation. In another embodiment of the invention, the entirecontainer can be a porous material with the formulation contained in thepores and the preservative incorporated into the polymer providing thepreservative effect.

In another embodiment of the invention, the surface of the device fromwhich the drops ultimately elute and the surface of the pores in theplug or the spherical particles in the plug can incorporate additionalpreservative, either through adsorption or by attachment or otherwise beincorporated as particles to ensure that any liquid left in the poresdoes not promote growth of microorganisms. As an example, the plug canbe pre-loaded with BAK at a suitable concentration to ensure that anymicroorganism that is trapped in the plug does not grow over time. Inanother embodiment, other antimicrobial particles, such as, silverparticles, can be incorporated into the plug to achieve the preservativeaction.

Typical eye drop dispensing systems employ a similar basic design. Aplastic bottle is elastic such that the application of force by thefingers pressing on the bottle leads to deformation that compresses theair in the bottle to impose an increase in pressure on the liquid, whichleads to drop creation at the tip. The flow of liquid out of the bottleresults in an increase of the gas phase volume and a decrease inpressure. The pressure needed for the drop creation must exceed theYoung Laplace pressure during drop creation, which is about 2σ/R_(d)where σ is the surface tension and R_(d) is the radius of the drop.Estimating R_(d)˜0.5 mm based on a drop volume of 30 μL, and usingsurface tension of water for σ, gives a value of about 100 Pa for theYoung Laplace pressure. Assuming an ideal gas law, to achieve thispressure, the volume of the gas phase (ΔP) in the bottle must decreaseby the volume ΔV=ΔP/P*V, where P is the starting pressure in the bottle(1 atmosphere) and V is the volume of the air phase. Substitutingapproximate values of all parameters gives ΔV/V=0.1%, which means thatthe pressure applied by the hands must be sufficient to achieve a 0.1%decrease in the volume of the bottle. However, an additional ΔV equalingthe volume of the drop is required to compensate for the increase in thevolume of the gas phase due to the volume of liquid pushed out of thebottle. The volume of typical eye drops is about 30 μL, which is largerthan the 0.1% of the volume of the bottle. Thus, the volume reductionnecessary for dispensing the eye drop is approximately equal to thevolume of the drop itself. The pressure generated in the gas phase fromthis compression, estimated by the ideal gas law, where ΔV=30 μL, V=3mL, P=1 atm, indicates a minimum ΔP of about 0.01 atm=1000 Pa. Thisrepresents the minimum pressure needed to create the drop. Most subjectscan easily apply 5-10 times this pressure.

Drop dispensing is more complex in a plug containing device, accordingto an embodiment of the invention, due to the extra pressure required topush the fluid through the plug As a patient squeezes the bottle, theincreased pressure in the gas phase will push liquid through the plug.Initially, the entire pressure drop will occur across the plug becausethe drop has not yet formed. As the drop forms and its volume increases,the Young Laplace pressure increases, reducing the available pressuredrop for flow through the plug. The rate of liquid flow through the plugdepends on the applied pressure as well as the design parametersincluding the length, area, porosity, and hydraulic permeability. Theseparameters are required of a plug such that a subject can instill theeye drop from the bottle containing the plug without having to applyexcessive pressure while the plug removes the desired amount of thepreservative from each eye drop till the entire formulation is used. Itis not a trivial exercise to determine a desired pore size and hydraulicpermeability. A higher pore size and permeability facilitatesinstillation of the eye drops but reduces the time of transit throughthe plug and the available surface area, which reduces the mass ofpreservative removed. Other aspects of the plug performance depend onthe hydraulic permeability. For example, after the eye drop isinstilled, the subject stops squeezing the bottle. This creates a vacuuminside the bottle, which retracts the remaining fluid at the tip of thebottle. When the bottle contains the plug, the entire plug is full ofthe fluid after the eye drop is instilled. The vacuum inside the eyedrop bottle could such back the entire fluid from the plug but thatwould depend on the hydraulic permeability as well as the surfacetension and the contact angle of the formulation with the plug material.If the hydraulic permeability is not sufficiently large, the plugretains the formulation between two successive instillations. This isbeneficial to the instillation process, as the volume of fluid needed tobe transferred would simply be the drop volume. However, the tip must beproperly sealed to minimize evaporation from the fluid filled plug. Itis critical to ensure that the plug displays pores of the plug that is asterile environment as the preservative from the formulations would betaken up by the plug. Even with a very high hydraulic permeability, somefluid is potentially trapped, necessitating the plug designed tomaintain sterility, for example, by preloading the plug with BAK, or anantimicrobial coating, or by adding antimicrobial particles in the plug.With each drop instillation, the concentration of BAK in the plugincreases, thereby assuring the sterility of the plug.

Below is provided a mathematical model of the fluid flow through theplug and the BAK uptake that permits determination of physicalproperties displayed by a plug (pore size, hydraulic permeability, crosssectional area, length) that allow one to achieve the objectives of eyedrop instillation without a significant increase in the pressurerequired and permit BAK removal to the desired extend from the entireformulation. It should be understood that the model is for a simplifiedversion of the device, yet permits estimates on the design parameters toachieve the desired separation. Experiments would eventually be neededto optimize the device starting from the parameters suggested by themodel.

The pressure drop through the plug can be estimated by Darcy's Law:

$\begin{matrix}{q = {\frac{k}{\mu}\frac{\Delta \; P}{H}A}} & \lbrack 1\rbrack\end{matrix}$

where k is the hydraulic permeability of the material, L is the length,μ is fluid viscosity, ΔP is the pressure drop across the plug and A isthe cross-sectional area. The average flow rate through the plug is theratio of the volume of the drop V_(d) (=30 μL) and the time needed toform the drop τ. We want τ of about 3 s, which is comparable to the timetaken to form a drop with most commercial bottles. Consideration of thedrop forming mechanics leads to the following constraint:

$\begin{matrix}{\frac{V_{d}}{\tau} = {\frac{k}{\mu}\frac{\Delta \; P}{H}A}} & \lbrack 2\rbrack\end{matrix}$

Plugs, according to an embodiment of the invention, are designed toselectively remove nearly all preservative BAK without reducing theconcentration of the active pharmaceutical ingredient (API). The plugmust have sufficient capacity for absorbing the preservatives loaded inthe bottle where the interactions between the plug material and thepreservative must be sufficiently strong to eliminate any desorption.The kinetics of preservative uptake by the material of the plug is veryrapid, such that the time scale for binding is shorter than the timescale for flow of the formulation through the plug.

The macroporous gel can be modeled as a set of parallel pores of lengthL and radius R to address the fluid flow and mass transfer in themacroporous gel and determine a structure that can achieve separationgoal of removing >90% BAK with a fluid flow where no increase in thepressure is required to create the drops. The concentration in pore c(r,z, t) is a function of the radial position in the pore r, axial positionalong the plug z, and time t. The solution of the convection-diffusionequation for BAK transport in the pore requires establishing appropriateinitial and boundary conditions

$\begin{matrix}{{\frac{\partial c}{\partial t} + {{u(z)}\frac{\partial c}{\partial z}}} = {D\left\lbrack {\frac{\partial^{2}c}{\partial z^{2}} + {\frac{1}{r}\frac{\partial}{\partial r}\left( {r\frac{\partial C}{\partial r}} \right)}} \right\rbrack}} & \lbrack 3\rbrack\end{matrix}$

where the velocity through the pore is given by the Poiseuille flowprofile, i.e.,

$\begin{matrix}{{u(z)} = {2{{\langle u\rangle}\left\lbrack {1 - \left( \frac{r}{R} \right)^{2}} \right\rbrack}}} & \lbrack 4\rbrack\end{matrix}$

where <u> is the average velocity of the fluid through the gel. To solvethe above convection-diffusion equation, the boundary and initialconditions are:

$\begin{matrix}{{c\left( {r,{z = 0},1} \right)} = {c_{0} + {\frac{d}{\langle u\rangle}\frac{\partial c}{\partial z}\left( {r,{z = 0},t} \right)}}} & \lbrack 5\rbrack \\{\frac{\partial c}{\partial z} = {\left( {r,{z = L},t} \right) = 0}} & \lbrack 6\rbrack \\{{c\left( {{r = R},z,t} \right)} = 0} & \lbrack 7\rbrack \\{{\frac{\partial c}{\partial r}\left( {{r = 0},z,t} \right)} = 0} & \lbrack 8\rbrack \\{{c\left( {r,z,{t = 0}} \right)} = 0} & \lbrack 9\rbrack\end{matrix}$

where C₀ is the inlet (z=0) concentration of the solute. The boundaryconditions at the inlet (z=0) and the outlet (z=L) are the ‘close-end’boundary conditions commonly used for modeling mass transport in packedbeds. The zero derivative at r=0 arises from the symmetry orequivalently no sink condition, and the boundary condition at the poreboundary (r=R) assumes rapid adsorption of BAK to the pHEMA matrix. Theinitial condition assumes that the concentration of surfactant is zerobefore the BAK solution is pushed through.

The above model applies the following assumptions and simplifications:the swelling of the plug (if any) is neglected because in the shortduration of flow, about 3 s, which is the target time for dropformation; and rapid binding of BAK to the pHEMA matrix occurs at thepore boundary (r=R), which is consistent with the very high partitioncoefficient of BAK in pHEMA and the 100% removal of BAK in flowexperiments, as indicated in the Examples below. The partitioncoefficient is the ratio of adsorption and desorption rate constants,where very high values can be interpreted as rapid adsorption, witheffectively zero concentration at the pore boundary. An approximatesolution can be obtained by neglecting diffusive contribution to axialflux, because the convective term is much larger than the diffusionterm. This approximation allows the steady state equation in thesimplified form:

$\begin{matrix}{{{{\overset{\sim}{u}(r)}\frac{\partial c}{\partial\eta}} = {\frac{DL}{{\langle u\rangle}R^{2}}\left\lbrack {\frac{1}{\zeta}\frac{\partial}{\partial\zeta}\left( {\zeta \frac{\partial}{\partial\zeta}} \right)} \right\rbrack}}{where}{{\eta = \frac{z}{L}},{\zeta = {{\frac{r}{R}\mspace{14mu} {and}\mspace{14mu} {\overset{\sim}{u}(r)}} = {\frac{u}{\langle u\rangle}.}}}}} & \lbrack 10\rbrack\end{matrix}$

The dimensionless parameter

$\frac{DL}{{\langle u\rangle}R^{2}}$

is the ratio of the time required for the fluid to travel through theplug to the time for the BAK molecules to diffuse from the center of thepore to the boundary. When this dimensionless parameter is much smallerthan one, the concentration in the pore is equal to the inletconcentration because the fluid travels far too quickly and so moleculesdo not have adequate time to diffuse radially and adsorb. If theparameter

$\frac{DL}{{\langle u\rangle}R^{2}}$

is much larger than one, file concentration of BAK in the eluting fluidshould be zero because the molecules have sufficient time to diffuse inthe radial direction and adsorb on the pore walls. By substituting theaverage velocity from Darcy's law, the requirement for complete removalof BAK from the eluting drop gives the following constraint:

$\begin{matrix}{\frac{D\; \mu \; L^{2}}{k\; \Delta \; {PR}^{2}} > 1} & \lbrack 11\rbrack\end{matrix}$

This can be simplified by using the Carman-Kozeny equation that givesthe following relationship between the hydraulic permeability to thepore size:

$\begin{matrix}{k = \frac{ɛ\; R^{2}}{4f}} & \lbrack 12\rbrack\end{matrix}$

where f is the Kozeny factor, which depends weakly on the porosity (ε),f=3.4(1−ε)^(0.175). To simplify this analysis, a constant value of 3 isused for f By substituting this calculated k into the Equations 2 and 11with known values of various physical and transport properties (μ, D)and other parameters that are required to be fixed by the designcriteria (ΔP, τ, V_(d)), the following constraints result:

$\begin{matrix}{{\frac{{AR}^{2}}{L} = \frac{4\; \mu \; V_{d}}{\tau \; \Delta \; P\; ɛ}}{and}} & \lbrack 13\rbrack \\{\frac{R^{2}}{L} < \sqrt{\frac{12\; \mu \; D}{\Delta \; P\; ɛ}}} & \lbrack 14\rbrack\end{matrix}$

for the instillation of a single drop of the formulation. If multipledrops are instilled, the fraction of BAK removed in the plug willdecrease with volume instilled because of the saturation of the plugwith BAK. In these calculations, targeted removal is at least 90% BAKeven from the last drop instilled, which would lead to >95% onconsidering all the drops instilled. Higher removal fractions can easilybe integrated into the model to yield the new predictions. To achievethis target the partition coefficient of the plug must be sufficientlyhigh such that the equilibrium concentration in the solution after theuptake of the entire BAK from the formulation in the gel is less than10% of the initial BAK concentration. The concentration in the gel afteruptake of the entire BAK in the formulation is

$\begin{matrix}{\frac{V_{f}c_{0}}{{AL}\left( {1 - ɛ} \right)},} & \;\end{matrix}$

which adds the following constraint in the design:

$\begin{matrix}{\frac{V_{f}c_{0}}{{AL}\left( {1 - ɛ} \right)} < {0.1\mspace{14mu} {Kc}_{0}}} & \lbrack 15\rbrack\end{matrix}$

where V_(f) is the total volume of the formulation passing through thegel plug and K is the partition coefficient defined as the concentrationof BAK in the gel phase divided by the concentration of BAK in thesolution phase.

The values of all parameters used in the calculations are listed inTable 1, below, and the design constraints obtained from the model(Equations 13-15) are graphically presented in FIG. 3, as these plotspredict, for the design parameters in Table 1 for a plug with length,pore radius and the corresponding hydraulic permeability of the BAK,when the plug area is 78.5 mm². The actual length of the plug is notnecessary equal to L, but can be L/T, where T is the tortuosity andestimated to be 3 by viewing the filter plug as a packed bed ofnon-uniform spheres. The design constraints suggest that the plug areamust be at least 0.258 mm² and preferably larger. As a reasonabledesign, if the area equals 78.5 mm², L should be at least 0.33 cm long.Therefore, therefore, when the filter plug is 0.5 cm in length and 78.5mm² in area, the minimum hydraulic permeability of 0.13 Da or a porediameter of 4 μm is needed.

All of the design parameter estimates are based on integrating thedevice into commonly used eye drop bottles. By re-designing the bottles,the parameters can be altered to improve the device performance. Forexample, the pressure available for drop instillation could besignificantly increased by changing the material of the eye drop bottle.The area of the plug can be adjusted by changing the bottle tip design.

TABLE 1 Typical values of the parameters used in the design constraintsof Eq. 13 to 15 Parameters Values Size of typical eye drop (V_(d)) 30 μLTotal volume of solution in bottle (V_(f)) 5 mL Viscosity (μ) 1.0 cPDiffusivity (D) 10⁻⁹ m²/s Pressure drop (ΔP) 5000 Pa^(a) Typical time tocreate a drop (τ) 3 s Porosity (ε) 0.4 Partition coefficient (K) 100^(a)The value is the estimated typical pressure drop created during theprocess of applying an eye drop with eye drop bottle.The porous plug can be included in the package for removal of BAK fromcommercial formulation. For example, the porous plug can be used withthe commercially available glaucoma drugs: Betimol®, which is a clear,isotonic, phosphate buffered aqueous solution containing 0.25% or 0.5%of drug timolol as hemihydrate, 0.01% BAK, and having inactiveingredients that include monosodium and disodium phosphate to adjust pH(6.5-7.5); COSOPT®, which is an isotonic, buffered, slightly viscous,aqueous solution containing a combination of two glaucoma drugs 0.5%timolol, 2% dorzolamide, 0.0075% BAK, and inactive ingredients sodiumcitrate, hydroxyethyl cellulose, sodium hydroxide, and mannitol;XALATAN®, which is an isotonic, buffered aqueous solution of 0.005%latanoprost, 0.02% BAK, and inactive ingredients sodium chloride, sodiumdihydrogen phosphate, and disodium hydrogen phosphate; LUMIGAN® whichcontains bimatoprost 0.3 mg/m; 0.05 mg/mL BAK, and inactive ingredientssodium chloride, sodium phosphate, and citric acid; and TRAVATAN®, whichcontains travoprost 0.04 mg/mL, 0.15 mg/mL BAK, and inactive ingredientspolyoxyl 40 hydrogenated castor oil, tromethamine, boric acid, mannitol,edetate disodium, sodium hydroxide and/or hydrochloric acid. The plugcan also be incorporated into any of the rewetting drop formulations.The above list is a small subset of all ophthalmic drug formulationsthat can have the preservative removed by integrating the plug with thebottle. The device can be a separate entity that is attached to theformulation dispensing units through suitable connectors.

Although the plugs according to embodiments of the invention areeffective for the removal of BAK and other preservatives, the inventionis not so limited. Components other than preservatives that are requiredin the formulation but are not needed inside the body, such as, but notlimited to formulation stabilizers and anti-oxidants can be removed.Other fluids can be used where a preservative is selectively removedfrom a fluid composition. Fluids that can be dispersed from a containerthrough a preservative removing device include intravenous drugs, oraldrug solutions and suspensions, foods, beverages, fragrances, lotions,soaps, shampoos, or any other fluid that is to be ingested, contactedwith skin, wounds, orifices, or openings made to a body. Although asdisclosed herein, the exemplary preservative is BAK, other preservativescommonly dissolved in an aqueous based solution, emulsion, or suspensioncan be removed from a preservative removing device, adapted to remove adesired preservative.

Methods and Materials Preparation of Macroporous Poly(2-HydroxyethylMethacrylate) Hydrogel

Macroporous poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel wasprepared by mixing 4 mL of HEMA monomer, 400 μL of ethylene glycoldimethacrylate (EGDMA), 15 mmoles of sodium chloride, 10 mg ofdiphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) and 15 mL ofdeionized (DI) water with magnetic stirring for 20 min at 900 rpm. Themixture was deoxygenated by bubbling pure nitrogen through the mixturefor 30 min. The deoxygenated mixture was poured into a 55×17 mm(diameter×height) Pyrex® petri dish, covered to prevent significantevaporation, and irradiated with UV light for 40 min using a UVB-10transilluminator (ULTRA-LUM, INC, Carson, Calif., USA) with an intensityof 16.50 mW/cm² sharply peaked at 310 nm. After polymerization, themacroporous pHEMA gel was carefully removed from the petri dish andsoaked in 350 mL of DI water for 24 hours to extract unreactedcomponents. The DI water was replaced with fresh DI water every 24 hoursfor a consecutive of 7 days to thoroughly remove the unreactedcomponents as confirmed by measuring the UV-Vis spectra of the waterfrom the proximity of the gel during the 7 days of extraction where theUV-Vis absorbance was negligible. The synthesized macroporous gel wasthen storage in DI water. The SEM image of the synthesized pHEMAhydrogel has pore size of few microns, as shown in FIG. 4.

Measurement of Hydraulic Permeability of the Macroporous Gel by Packingin Syringe

To determine the pressure applied when a bottle is squeezed, a bottlewas held vertically with the exit pointed down and squeezed to determinethe mass of liquid that will elute or the number of drops that fall out.The pressure inside the gas phase created by the squeeze can then bedetermined as ΔV/V×P_(atm), where ΔV is the volume of liquid that elutesout, V is the volume of the gas inside the bottle, P_(atm) is theatmospheric pressure. This method provided an estimate of about 5000 Paas the pressure generated inside the eye drop bottle during the squeeze.This pressure is not the same as the applied pressure by the fingers.The force/pressure applied by the fingers squeezes the bottles, which inturn reduces the volume inside the bottle. That volume reduction leadsto the pressure increase. After determining the available pressure, anestimate of the velocity through the filter based on creation of a dropin about three seconds was carried out. As the required permeabilitydepends on the filter design, the estimates suggest that a hydraulicpermeability larger than about 0.1 Da will be adequate with permeabilityof about 1 Da or larger being more suitable for an eye drop device.Higher values are needed with more viscous solutions, such as wettingdrops. While 0.1 Da is adequate for drop dispensing, it is notsufficient for retraction of the fluid remaining in the filter after thedrop dispensing.

To test the hydraulic permeability of the matrix, the BAK removalmaterial was packed in a sterile syringe (SOFT-JECT®, 3 ml, Henke-SassWolf GmbH, Tuttling, Germany) of 1 cm in diameter. Two pieces of filterpapers (Qualitative 1, Whatman®, Maidstone, England) were placed in thesyringe to prevent the packed material from leaking out due to theapplied pressure. To make the BAK removal material uniformly packed, 2.5mL of DI water was pushed through the syringe with high pressure eachtime and repeated at least 5 times. The syringe packed with BAK removalmaterial was used for the hydraulic permeability measurement using thesetup was shown in FIG. 5. A beaker was placed immediately below thesyringe to collect the outlet solution. The syringe was filled with 2.5mL of PBS (viscosity=1.00±0.05 cP) and a 1.28 kg (1.6×10⁵ Pa) weight wasplaced on the tray to create the pressure drop across the packing. Theprocess was timed using a stopwatch that started with placing the weighton the tray to the time the last drop dropped into the beaker. Theweight of the collected PBS solution in the beaker was measured tocalculate the flow rate through the filter material. The hydraulicpermeability coefficient was then calculated by Darcy's law, Equation 1,above.

The hydraulic permeability of the macroporous pHEMA gel prepared asindicated above, was measured 10 times for each of 12 samples, with theresulting permeability shown in FIG. 6. As shown in FIG. 6, thehydraulic permeability of the gel slightly decreased over themeasurement, although the trend was not significant. This was due to thehigh pressure exerting on the gel in each run made the gel pack moretightly. The overall average of the hydraulic permeability is 0.025Darcy.

Performance Testing of the Macroporous pHEMA Gel as BAK Removal Filter

The selectivity of BAK removal was tested with four common ophthalmicdrugs including timolol maleate (hydrophilic, medication for glaucoma),dorzolamide hydrochloride (hydrophilic, medication for glaucoma),latanoprost (hydrophobic, medication for glaucoma) and dexamethasone(hydrophobic, medication for infection or eye injuries). The four drugswere dissolved in PBS and mixed with BAK individually. The prepareddrug/BAK mixture concentration is summarized in Table 2, below. Themacroporous pHEMA hydrogel prepared as above, was used as the BAKremoval filter and packed into the syringe, as described above. Theexperimental setup is that of FIG. 3. A 2.5 mL of drug/BAK solution wasplaced in the syringe and forced through the filter plug by the pressuredrop created by the weight (1.28 kg) on the tray. The solution thatpassed through the filter plug was collected and the concentration ofdrug/BAK was determined by measuring the UV-Vis spectra. The detectedwavelength range for each drug/BAK mixture is summarized in Table 2,below. The measured UV spectrum was a linear combination of the testingdrug and BAK and thus, the individual concentration of the drug and BAKcould be determined by applying a least square fit method as describedin Kim et al. Int. J. Pharm., 2008Apr. 2; 353(1-2):205-22 which wasvalidated by comparison to standard mixture solutions of drug and BAK.The same experiment procedure was repeated 10 times for each macroporouspHEMA gel plug. The test solutions contained 0.012 wt % BAK, which waswithin the normal BAK concentrations, 0.004 wt % to 0.025 wt %, used incommercial eye drops. The drug concentration was adjusted accordingly tothe BAK concentration so that the measured UV-Vis spectrum would besignificantly different if 100% of the BAK was removed. To be morespecific, if 100% of BAK was removed, the UV absorbance at 261 nm, whichis the maximum of the BAK spectrum, would decrease by about 50%. The BAKconcentration in latanoprost/BAK solution was reduced to 0.003 wt %,which is within the detection limit. The concentration of latanoprostwas adjusted such that the UV absorbance in the range of 210-220 nmwould decrease by about 50% if all BAK was removed.

FIGS. 7-10 show the percentages of BAK and drug that are absorbed afterthe mixture solution was passed through the macroporous pHEMA gel plug.As shown in the figures, 100% of BAK was removed by the hydrogel in thefirst run regardless of the drug in the mixture. The BAK removalpercentages decreased after each subsequent pass due to the gel becomingslowly saturated with BAK and removal decreased to 70-80% at the 10^(th)pass. For hydrophilic drugs, such as timolol (FIG. 7) and dorzolamide(FIG. 8), the drug adsorption percentages was low, and remained about 5%after the first pass through the 10^(th) pass. Macroporous pHEMAhydrogel exhibited an excellent BAK removal efficiency with littlehydrophilic drug uptake. However, the drug absorption percentages wereas high as 90 and 65% in the first run for hydrophobic drugs oflatanoprost (FIG. 9) and dexamethasone (FIG. 10), respectively. Theabsorption percentage decreased rapidly to 30% and 10% by the 10^(th)pass through the gel for latanoprost and dexamethasone, respectively;which suggested that one may pre-equilibrate the macroporous pHEMA gelwith drug solution to allow the gel to absorb BAK without uptake of anyadditional drug.

TABLE 2 Summary of the drug/BAK mixture concentrations as prepared andthe UV-Vis wavelength detection range used for testing the separationselectivity Timolol Dorzolamide Latanoprost Dexamethasone Drug 0.010.005 0.03 0.005 concentration (mg/ml) BAK 0.12 0.12  0.03 0.12 concentration (mg/ml) Wavelength of 261-309 231-279 210-220 237-279UV-Vis (nm) ^(a)PBS was used as the solventMeasurement of Partition Coefficient of Timolol Maleate, DorzolamideHydrochloride, Latanoprost, Dexamethasone and BAK in Macroporous pHEMAHydrogel

As indicated above, the absorption percentage of hydrophilic drug issmall, whereas a significant amount of hydrophobic drug is uptake by themacroporous pHEMA gel. The high affinity of hydrophobic drug to the gelcan be determined by measuring the partition coefficient of the drug inthe gel. To measure the partition coefficient, a piece of macroporouspHEMA gel of 250 mg was soaked in 12 mL of drug or BAK solution. PBS wasused as the solvent and the prepared concentrations of the drug and BAKsolutions are summarized in Table 3, below. After 15 days of soaking,the concentration of the drug or BAK solution was measured by usingUV-Vis spectrophotometry. The drug or BAK concentration after soakingindicated the amount of drug or BAK in reference to the initialconcentration is that absorbed into the gel. Partition coefficientscalculated from the concentration change is summarized in Table 4,below. The partition coefficient of timolol and dorzolamide inmacroporous pHEMA gel is roughly 15 times smaller than that of BAK,which is excellent separation efficiency. In contrast, the partitioncoefficients of latanoprost and BAK are pretty high and the separationefficiency of the gel for this mixture is poor.

TABLE 3 Concentration of drug and BAK in PBS solutions. Dexa- TimololDorzolamide Latanoprost methasone BAK Concentration 0.08 0.07 0.04 0.072.4 (mg/ml)

TABLE 4 Partition coefficient of drugs and BAK in macroporous pHEMA gel.Dexa- Timolol Dorzolamide Latanoprost methasone BAK Partition 6.49 ±7.59 ± 0.56 90.18 ± 33.53 ± 1.31 101.61 ± co- 0.37 1.36 11.51efficient^(a) ^(a)as mean ± SD with n = 3Performance Testing of the Macroporous pHEMA Particles for BAK Removal

Macroporous pHEMA hydrogel as prepared above was dried in the oven of80° C. and crushed into particles and the particles packed into thesyringe. Two pieces of filter papers were placed at the bottom of thesyringe to prevent the packing particles from leaking out. Particlesfacilitate packing of a hydrogel into the neck of the eye drop bottlewhere packing is amenable to high-throughput industrial scale loading ofgel filters. To evaluate the performance of the pHEMA particles, thehydraulic permeability and the selectivity of separation of BAK fromtimolol, dorzolamide, latanoprost and dexamethasone were measured withthe same experiment setup shown in FIG. 5. The prepared concentrationsof the 4 different drug/BAK mixtures were summarized in Table 2, above.The syringe was filled with 2.5 mL of drug/BAK solution and a beaker wasplaced below the syringe to collect the outlet solution. A 1.28 kg(1.6×10⁵ Pa) weight was placed on the tray to create the pressure dropand force the solution through the packing of pHEMA particles. Theprocess was timed using a stopwatch from the moment of placing theweight to the last drop entered the beaker. The weight of the collectedsolution in the beaker was measured to calculate the flow rate throughthe pHEMA particles. The hydraulic permeability was calculated byDarcy's law (Eq. 1) where the cross section area of the syringe was0.785 cm² and the height of the packed pHEMA particles was 5 mm. Becausethe concentration of the drug/BAK solution was fairly dilute, theviscosity of the solution was approximated that of pure PBS (1.00±0.05cP). The UV spectrum of the collected solution was measured and theindividual concentration of the drug and BAK were determined by a leastsquare fit method as described in Kim et al., Int. J. Pharm. 2008Apr. 2;353(1-2):205-22.

BAK has a high partition coefficient of roughly 100 in macroporous pHEMAhydrogel as indicated in Table 4, above. Surprisingly, the BAK removalpercentage decreased at an early stage as more drug/BAK solution passedthrough the macroporous pHEMA hydrogel, as indicated in FIGS. 7-10. Totest if the early decrease was due to saturation of the BAK at thesurface of the gel due to slow diffusion into the gel particlesexperimental runs were separated by 24 hours and only 2.5 mL of drug/BAKsolution was passed through the particulate plug each day allowing thediffusion of the BAK from the surface into the interior of the gelparticles. The hydraulic permeability and selectivity of separation weremeasured each time the drug/BAK solution passed through the pHEMAparticles. After each measurement, the bottom outlet of the syringe wassealed with parafilm to prevent dehydration of the packed pHEMAparticles in the manner equivalent to sealing the eye drop bottle withthe cap after use.

FIGS. 11-14 show the percentages of BAK and drug absorbed from thesolution after each pass through the pHEMA particulate plug. Asillustrated in FIGS. 11-14, nearly 100% of containing BAK was removed bythe crushed macroporous pHEMA particles for all 10 passes, regardless ofthe drug in the solution. Hence a 24 hours period between passes allowsthe dilution of the surface BAK concentration. The time required forequilibration of the gel particles is much less than 24 hours in apractical eye drop application. The pHEMA particles should be able toremove 100% of BAK from the entire content of a typical eye dropcontainer if use by a single patient in a typical prescribed manner.FIGS. 11-14 display an excellent separation efficiency of BAK from alltested drugs. The pHEMA particles used for latanoprost/BAK anddexamethasone/BAK selectivity were equilibrated with the correspondingdrug solution in advanced by simply soaking the pHEMA particles inlatanoprost/PBS or dexamethasone/PBS solution. Therefore, only a verysmall portion of latanoprost and dexamethasone were absorbed as thepre-saturation of the drug in the particles, allowed passage of the drugwithout absorption even though the BAK was effectively partitioned intothe pHEMA hydrogel.

The hydraulic permeability of the crushed macroporous pHEMA particles isplotted in FIG. 15, where a significant decrease of the averagepermeability from 0.025 Darcy on day 1 to 0.004 Darcy on day 10. Thisresulted from the high pressure exerting on the particles for each passcausing the particles to pack more extensively, reducing the volumewithin the plug for solution flow. For commercial application it isimportant to prevent decrease in hydraulic permeability with use, whichcan be achieved by increasing the rigidity of the particles, forexample, by increasing the crosslinking density of the gel.

Preparation of Macroporous HEMA-Methacrylic Acid (MAA) CopolymerHydrogel

As shown in FIGS. 9, 10, 13 and 14, because of the high partitioncoefficient of hydrophobic drugs in macroporous pHEMA hydrogel, asindicated in Table 4, a significant amount of latanoprost anddexamethasone were removed after passing the drug/BAK solution throughthe hydrogel. Hydrophilic content of the hydrogel can be increased byaddition of comonomers to the polymer, such as, dimethyl acrylamide(DMA), methacrylic acid (MAA), or any other biocompatible, high watercontent polymer which can result in less affinity for the hydrogel tothe hydrophobic drugs.

To prepare the macroporous HEMA-co-MAA copolymer hydrogel, 3.2 mL ofHEMA, 0.4 mL of EGDMA, 0.8 mL of MAA, 4 mmole of sodium chloride, 15 mLof deionized water and 10 mg of TPO were mixed in a glass vial followedby the same steps carried out to form hydrogel, as described above. Theresulting hydrogel was subsequently packed into a syringe as describedabove. A 2.5 mL portion of dexamethasone/BAK solution was passed throughthe hydrogel to test separation selectivity, and the step of passing wasrepeated three times on the same hydrogel plug. The height of thepacking hydrogel in the syringe was 5 mm. The concentration of thedexamethasone and BAK mixture was 0.005 and 0.12 mg/ml, respectively,and PBS was used as solvent. The results were shown in FIG. 16. Asopposed to the macroporous pHEMA hydrogel, as indicated in FIG. 10, thepercentage of dexamethasone being absorbed was reduced from 65% to 45%in the first pass.

Alternately, the macroporous pHEMA hydrogel was prepared by theprocedure above followed by soaked into 5%, 2% and 1% MAA solution for 3hours. DI water was used as the solvent to prepare the MAA solution. A10 mg quantity of potassium persulfate was added to the solution as athermal initiator. The hydrogel and the solution were placed in an 80°C. oven overnight. The MAA treated pHEMA hydrogel was taken out of thevial and washed with large quantity of DI water to remove unreactedcomponents. The hydrogel was packed into the syringe as BAK removalfilter and its separation efficiency of BAK from dexamethasone wastested in the same manner as the copolymer. The hydraulic permeabilityof the hydrogel copolymerized with 5% of MAA solution was too low topass solution through the gel. The separation efficiencies of thehydrogels from 1 and 2% of MAA solution are similar. The result of thehydrogel treated with 1% MAA is shown in FIG. 17. Nearly 100% of BAK wasremoved in the 3 consecutive runs, while the percentage of dexamethasonebeing absorbed was diminished to 17% in the first run.

Preparation of pHEMA Particles by Heat-Initiated Polymerization UsingEGDMA as the Cross-Linker

To a mixture of 1.2 mL of HEMA, 0.3 mL of EGDMA, 12 mL of DI water, and600 mg of magnesium oxide, 10 mg of benzoyl peroxide was added in aglass vial and the contents magnetic stirring for 20 minutes at 900 rpm.The presence of magnesium oxide caused the mixture to phase separated.Small globules containing HEMA monomer and EGDMA was formed bycontinuously stirring the system at high rpm. The mixture wasdeoxygenated with pure nitrogen for 30 min. The mixture was warmed usinga water bath at 70° C. for 18 hours with continuous stirring at 900 rpmto retain small globules that polymerize into individual pHEMAparticles. After polymerization, pHEMA particles were separated from themixture solution by vacuum filtration method and washed with a largequantity of DI water to remove unreacted monomers and other impuritiesand dried in an oven of 80° C.

The SEM image of the synthesized pHEMA particles was shown in FIG. 18.The pHEMA particles have wrinkled, “brain-like” surfaces with a largesize range from 10 to 300 μm. The particles were packed in the prototypebottle shown in FIG. 19.

Measurement of Hydraulic Permeability of BAK Filter Packed in an EyeDrop Bottle Prototype

FIG. 19 shows a design for an eye drop bottle prototype, which was usedto measure the hydraulic permeability of the BAK removal filter packedin the tip. The bottle can be any commercially available eye dropbottle. A section of rigid plastic tube was attached to the tip of theeye drop bottle and the connection part of the bottle to the plastictube was sealed to prevent leakage. The plastic tube was transparent.Two layers of filter papers are placed at the two ends of the BAK filterplug to prevent the filter plug from being displaced in eitherdirection.

To measure the hydraulic permeability of the packed BAK removal filter,the eye drop bottle was turned upside down and squeezed by fingers tocreate a pressure drop that forced the eye drop solution into theplastic tube section. Once the applied pressure was removed, thesolution flowed back into the bottle. By measuring the flow rate of thesolution returning into the bottle, Darcy's law (Eq. 1) was used tocalculate the hydraulic permeability of the BAK filter. The exactpressure drop across the filter plug was determined in the followingmanner. Since the temperature change is negligible and the mass of thegas in the eye drop bottle remains constant before and after thesqueezing, we know from the ideal gas law that

$\begin{matrix}{{P_{0}V_{0}} = {{{P_{f}\left( {V_{0} + {\Delta \; V}} \right)}\mspace{14mu} {or}\mspace{14mu} P_{f}} = \frac{P_{0}V_{0}}{V_{0} + {\Delta \; V}}}} & {{Eq}.\mspace{11mu} 16}\end{matrix}$

where P₀ is the pressure in the eye drop bottle before the bottle issqueezed which also equals to atmospheric pressure, P_(f) is thepressure in the bottle after the bottle is squeezed, V₀ is the gasvolume in the bottle before the bottle is squeezed and ΔV is the volumeof solution being pushed out of the bottle. The pressure drop (ΔP) thatpushes the solution back would be

$\begin{matrix}{{\Delta \; {P(t)}} = {{P_{0} - {P_{f}(t)}} = \frac{P_{0}\left\lbrack {{\Delta \; V} - {V^{\prime}(t)}} \right\rbrack}{V_{0} + \left\lbrack {{\Delta \; V} - {V^{\prime}(t)}} \right\rbrack}}} & {{Eq}.\mspace{11mu} 17}\end{matrix}$

where V′ is the volume of the solution that has already passed throughthe filter and got back into the bottle. Note that the V′ and ΔP is afunction of time. By doing a simple order of magnitude analysis, theeffect of gravity force on the solution is sufficiently small than theeffect of a pressure drop and hence the influence from gravity isnegligible. One can, therefore, rewrite Darcy's law (Eq. 1) as:

$\begin{matrix}{{\frac{{dV}^{\prime}}{dt} = {\frac{{kAP}_{0}}{\mu \; h}\frac{{\Delta \; V} - V^{\prime}}{V_{0} + \left( {{\Delta \; V} - V^{\prime}} \right)}}},} & {{Eq}.\mspace{11mu} 18}\end{matrix}$

where k is the hydraulic permeability, μ is the viscosity of thesolution and h is the length of the filter plug. This is an ODE equationand V′ can be easily solved as a function of time. The equation can befurther simplified because V′ is much smaller than V₀+ΔV and thus Eq. 18becomes:

$\begin{matrix}{\frac{{dV}^{\prime}}{dt} = {\frac{{kAP}_{0}}{\mu \; h}\frac{{\Delta \; V} - V^{\prime}}{V_{0} + {\Delta \; V}}}} & {{Eq}.\mspace{11mu} 19}\end{matrix}$

with the initial condition of

t=0,V′=0.  Eq. 20

The solution to Eq. 19 and 20 is

$\begin{matrix}{V^{\prime} = {\Delta \; {{V\left\lbrack {1 - {\exp \left( {{- k}\frac{{AP}_{0}}{\mu \; {h\left( {V_{0} + {\Delta \; V}} \right)}}t} \right)}} \right\rbrack}.}}} & {{Eq}.\mspace{11mu} 21}\end{matrix}$

The eye drops bottle Systane® was used. The weight of the empty bottlewas measured to be roughly 5.5 grams. The bottle was then filled withwater and the total mass was 22.5 grams. Subsequently, 12 grams of waterwas squeezed from the bottle so that V₀ would be roughly 12 mL and thewater left in the bottle is roughly 5 mL. The filter material preparedby thermal initiation, as disclosed above, was used and the packinglength is 8 mm. The cross area of the plastic tube was 0.0314 cm² andthe viscosity of water at 20° C. is about 1.002×10⁻³ Pa·s.

An eye drop bottle was turned upside down to squeeze out 1.5 mL of water(ΔV). This relatively large volume, 1.5 mL, of water creates asufficient pressure drop to draw the water back at a reasonable flowrate allowing the simplification from Eq. 18 to Eq. 19 with sufficientaccuracy. The process of water flowing back into the eye drop bottle wasfilmed where from V′ as a function of time was analyzed. The above model(Eq. 21) was used to fit the experiment data (V′ vs. t) to determine thehydraulic permeability (k) by using the function “fminsearch” inMATLAB®. The fit to the model is reasonably good and the result is shownin FIG. 20. The hydraulic permeability was determined to be 0.0459Darcy.

Selective BAK Removal by Crushed Macroporous pHEMA Particles Integratedinto Eye Drop Bottle Prototype

The crushed macroporous pHEMA particles were prepared as describedabove. The particles were packed in the eye drop bottle prototype (FIG.19) to test its selectivity of separation of BAK from latanoprost. Theconcentration of latanoprost and BAK prepared for the testing were both0.03 mg/mL with PBS as the solvent. The drug/BAK solution was injectedinto the prototype bottle with a syringe. A clip was clipping on thebottle to create a constant pressure drop across the packing pHEMAparticles of 8 mm in length. A volume of 1.5 mL of the drug/BAK solutionwas passed through the filter by squeezing the bottle. The UV spectrumof the outlet solution was measured and the individual concentration ofthe drug and BAK was determined by a least square fit method asdescribed in Kim et al., Int. J. Pharm., 2008Apr. 2; 353(1-2):205-22.The tip of the prototype bottle was sealed with parafilm. After 24hours, another 1.5 mL drug/BAK solution was removed through the samefilter and again to measure the concentrations of the drug and BAK. Thestep was repeated 10 times over a total of 10 days.

FIG. 21 showed the percentages of BAK and latanoprost absorbed after themixture solution flowed through the pHEMA particles. The pHEMA particleshad been pre-equilibrated with the latanoprost as described above tosuppress the amount of drug absorbed. Nearly 100% of containing BAK wasremoved by the particles in all 10 runs.

Selective BAK Removal by pHEMA Particles Prepared by UV-InitiatedPolymerization Using EGDMA as the Cross Linker Integrated into Eye DropBottle Prototype

As shown in FIG. 15, the plug of crushed macroporous pHEMA hydrogel hasa very low hydraulic permeability. Alternatively, pHEMA particles wereprepared photo chemically where 1.2 mL of HEMA, 0.3 mL of EGDMA, 12 mLof DI water, 900 mg of sodium chloride and 10 mg of TPO initiator weremixed in a glass vial and magnetic stirring for 20 minutes at 900 rpm.The sodium chloride promoted phase separation of the mixture. Smallglobules containing HEMA monomer and EGDMA was formed by continuouslystirring the system at high rpm. The mixture was then deoxygenated usingpure nitrogen for 30 min. The mixture was poured into a 55×17 mm(diameter×height) Pyrex® petri dish and irradiated with UV light for 2hours by a UVB-10 transilluminator (ULTRA•LUM, INC, Carson, Calif., USA)with an intensity of 16.50 mW/cm² sharply peaked at 310 nm. During theUV curing, the mixture was continuously stirred by a 35×6 mm magneticstirring bar at 70 rpm so that the small globules would remain separatedand polymerize into individual pHEMA particles. In addition, the petridish was covered to avoid water evaporation and oxygenation. After thepolymerization, the pHEMA particles were separated from the solution byvacuum filtration method and washed with a large quantity of DI water toremove the unreacted monomers and other impurities. The particles werethen dried in an oven of 80° C.

An SEM image of the synthesized pHEMA particles is shown in FIG. 20. ThepHEMA particle size has a wide range, from 10 to as large as 200 μm,which have a spherical shape with a smooth surface. The synthesizedparticles were packed in the prototype bottle and tested for theirselectivity of separation of BAK from timolol. The length of the plug ofthe packed particles was 8 mm. The timolol and BAK concentrationprepared for the testing were 0.01 and 0.12 mg/mL, respectively, withPBS as the solvent. The drug/BAK solution was injected into theprototype bottle with a syringe. A clip was applied to the bottle toimpose a constant pressure drop. A 1.5 mL aliquot of the drug/BAKsolution was forced through the filter by squeezing the bottle. The UVspectrum of the outlet solution was measured and the concentrations ofthe drug and BAK were determined by the least square fit methoddescribed in Kim et al., Int. J. Pharm., 2008Apr. 2; 353(1-2):205-22.Five samples were successively removed through the plug.

FIG. 23 showed the percentages of BAK and timolol that absorbed in thefilter from the mixture after each aliquot was passed through the pHEMAparticles. Roughly 50% of BAK was removed by the pHEMA particles in thefirst pass, while only 30% was removed on the 5^(th) run. Only about1.5% of the timolol were removed by the pHEMA particles in each of the 5runs.

Performance Testing of pHEMA Particles Prepared by Heat-InitiatedPolymerization as BAK Removal Filter Integrated into Eye Drop BottlePrototype

FIG. 24 indicates the percentages of BAK and timolol that were absorbedafter the mixture in solution was passed through the heat-initiatedpHEMA particles, shown in FIG. 18. As shown in FIG. 24, nearly 100% ofthe BAK was removed by the particles in each of 10 passes that werecarried out successively. About 17% of timolol was removed in the 1strun while the amount removed reduced to about 3% in the 10^(th) run. Thehydraulic permeability of the packing particles is 0.0459 Darcy whichwas measured as described, above. Due to the increased particle size,the hydraulic permeability is significantly improved when compared tothat of the particles prepared by crushing the macroporous pHEMAhydrogel. The size of the particles prepared by UV or heat-initiatedpolymerization is similar. However, in contrast to the particles withthe smooth surface prepared by UV-initiated polymerization, as shown inFIG. 22, the heat initiated polymerization method produces wrinkled,“brain-like” structures, as shown in FIG. 18, which provides a largesurface area for absorbing BAK, allowing a much higher BAK removalefficiency. Selectivity for the separation of BAK from timolol wastested. The timolol and BAK concentration prepared for the testing were0.01 and 0.12 mg/mL, respectively, with PBS as the solvent. The drug/BAKsolution was injected into the prototype bottle with a syringe. A clipwas clipping on the bottle to create a constant pressure drop across the8 mm height of packing. 1.5 mL of the drug/BAK solution was pushedthrough the filter by squeezing the bottle. The UV spectrum of theoutlet solution was measured and the individual concentration of thedrug and BAK was determined by a least square fit method as described inKim et al., Int. J. Pharm., 2008Apr. 2; 353(1-2):205-22. This step wasrepeated 10 times immediately on the same filter sample without waiting.

Performance Testing of pHEMA Particles Prepared by UsingTrimethylolpropane Ethoxylate Triacrylate as Cross-Linker

More rigid and larger size particles create a larger void space forfluid to flow and improved hydraulic permeability. If the particleshydrate significantly, the void volume and hydraulic permeability willchange significantly depending on the degree of hydration. This would beundesirable because the plug is drug at the time of the instillation ofthe first drop but then could be partially or fully hydrated forsubsequent instillations, depending on whether the plug retains thefluid in the interim time between successive instillations.Trimethylolpropane ethoxylate triacrylate (SR454HP or SR9035) was addedto the pHEMA particles formulation as cross-linker. pHEMA particles wereprepared photochemically where 1.4 mL of HEMA, 0.1 mL oftrimethylolpropane ethoxylate triacrylate, 12 mL of DI water, and 10 μLof 2-hydroxy-2-methyl-1-phenyl-propan-1-one were mixed in a glass vialand magnetic stirring for 20 minutes at 900 rpm. The mixture wasdeoxygenated using pure nitrogen for 30 min. The mixture was poured intoa 55×17 mm (diameter×height) Pyrex® petri dish and irradiated with UVlight for 2 hours by a UVB-10 transilluminator with an intensity of16.50 mW/cm² sharply peaked at 310 nm. During the UV curing, the mixturewas continuously stirred by a 35×6 mm magnetic stirring bar at 70 rpm.In addition, the petri dish was covered to avoid water evaporation andoxygenation. After polymerization, the pHEMA gel were separated from thesolution by vacuum filtration method and washed with a large quantity ofDI water to remove the unreacted monomers and other impurities. ThepHEMA gel was then dried in an oven of 80° C. and crushed into particlesin a mortar.

The SEM image of the synthesized pHEMA particles is shown in FIG. 25.The pHEMA particle size has a wide range, from 30 to as large as 900 μm,and has a highly irregular shape. The synthesized particles were packedin the prototype bottle and tested for their hydraulic permeability, asdescribed above. The length of the plug of the packed particles was 1.8cm. The measured hydraulic permeability of dried particles preparedusing SR454HP as cross-linker is 4.95±0.91 Da (n=3); whereas thehydraulic permeability of hydrated particles reduced to 2.34±0.39 Da(n=3). The measured hydraulic permeability of dried particles preparedusing SR9035 as cross-linker is 4.10±0.26 Da (n=3); whereas thehydraulic permeability of hydrated particles reduced to 1.22±0.33 Da(n=3). Compared to the particles prepared by other formulation, asdescribed above, the hydraulic permeability significantly increased morethan 25 times, which particles are thus suitable for removing BAK fromformulation that has a high viscosity, such as carboxymethyl cellulose(CMC) lubricant eye drops. The high permeability likely arises from thelarge size and the irregular shape. The irregular shape with sharp edgescan prevent drainage of the fluid from the plug back into the containerafter the applied pressure is removed and thus keeping the plughydrated. It is important to minimize evaporation from the bottle. Whenwater evaporation is critical, a layer of a hydrophobic particles couldbe placed at the top of the BAK removing particles as an extra barrier.

The partition coefficients of timolol, CMC and BAK in pHEMA particlesprepared using SR9035 as cross-linker was measured. pHEMA particles (100mg) were soaked in 3.5 mL of timolol, CMC and BAK solution, whichconcentration was 0.08 mg/mL, 0.5% and 2.4 mg/mL, respectively. After 9days of soaking, the concentration of the drug or BAK solution wasmeasured by using UV-Vis spectrophotometry. The drug or BAKconcentration after soaking indicated the amount of drug or BAK thatabsorbed into the gel relative to the initial concentration. Partitioncoefficients calculated from the concentration change are summarized inTable 5, below. The partition coefficient of timolol and CMC in pHEMAparticles is much smaller than that of BAK, and should have excellentseparation efficiency.

TABLE 5 Partition coefficient of drugs and BAK in pHEMA particlesprepared by using SR9035 as cross-linker. Timolol CMC BAK Partitioncoefficient 5.59 ± 0.13^(a) <1 ~300 ^(a)as mean ± SD with n = 3

Selectivity of the separation of BAK from timolol was measured. Thetimolol and BAK concentration were 0.01 and 0.12 mg/mL, respectively,with PBS as the solvent. A constant pressure drop was applied across thepacked particles to maintain a constant flow rate through the plug. A1.5 mL aliquot of the drug/BAK solution was forced through the filter bysqueezing the bottle. The UV spectrum of the outlet solution wasmeasured and the concentrations of the drug and BAK were determined bythe least square fit method described in Kim et al., Int. J. Pharm.,2008Apr. 2; 353(1-2):205-22. The tip of the prototype bottle was sealedwith parafilm. After 24 hours, another 1.5 mL drug/BAK solution wasremoved through the same filter and again to measure the concentrationsof the drug and BAK. The step was repeated 10 times over a total of 10days.

FIGS. 26 and 27 showed the percentages of BAK and timolol that absorbedin the filter from the mixture after each aliquot was passed through thepHEMA particles prepared by using SR454HP and SR9035 as cross-linker,respectively. In FIG. 26, nearly 100% of the BAK was removed by theparticles in the first 3-5^(th) runs, but reduced to about 90% after the5^(th) run. About 18% of timolol was removed in the 1^(st) run while theamount removed became negligible in the 10^(th) run. In FIG. 27, pHEMAparticles prepared by using SR9035 as cross-linker showed a slightlybetter BAK removal capacity, where nearly 100% of the BAK was removed bythe particles in the first 6^(th) runs, but reduced to about 95% in the10^(th) run. About 25% of timolol was removed in the 1^(st) run whilethe amount removed became negligible after the 5^(th) run.

BAK Removal from Bimatoprost Solutions by Crushed Macroporous pHEMA-MMAParticles Integrated into Eye Drop Bottle Prototype

Gels of pHEMA-MMA were synthesized using 2 mL monomer solution, 2.7 mLof water, 10 μL of ethylene glycol dimethacrylate as crosslinker, and 6mg of Darocur TPO as a photoinitiator. The monomer solution haddifferent fractions of HEMA and MAA (i.e. 60% MAA would be 1.2 mL MAAand 0.8 mL HEMA). Gels were cured under UV light in 100 micron thickmolds and subsequently cut into pieces approximately 50 mg in mass. Somegels were loaded with BAK to give a 3× (or 300 ppm) initialconcentration and placed into 3 mL solution (either 0.025%Bimatoprost/PBS 1× or 0.2% BAK/PBS). The concentrations in solution weremeasured using UV-vis spectrophotometry. Upon achieving equilibrium, thegels were placed in 3 mL blank PBS, and release was monitored by UV-visspectrophotometry. Uptake and release equilibrium concentrations wereused to calculate partition coefficients. FIGS. 28 and 29 are plots ofthe partition coefficient for Bimatoprost and BAK for various gelcopolymer compositions. Clearly at higher MAA concentrations theBimatoprost tends to remain in solution, whereas BAK strongly partitionsinto the gel for all gel copolymer compositions.

Bimatoprost Concentration in Eluting Drops from a Bottle Packed with0.06 g of p-HEMA Particles

A gel was prepared from 1.4 ml of HEMA monomer, 0.1 ml of a cross linker(SR9035), 12 ml of deionized (DI) water, and 20 μl of photo initiatorDarocur® 1173 that were mixed in a 20-ml vial and put under UV light andconstant stirring to produce particles. A filter tip was prepared byinserting in a layer of 11 micron pore size filter paper and 0.06 g ofp-HEMA particles were placed into the filter tip. The particles werecompressed and then covered with filter cloth. The bottle was thenfilled with 5 mL of 0.01% bimatoprost/PBS 1×. A drop was dosed out andmeasured using UV-vis spectrophotometry and compared to a drop that didnot pass through a filter to determine percent uptake of drug and BAK.As illustrated in FIG. 30, after dispensing of 14 drops, little or noadditional Bimatoprost absorbed in the gel particles.

Bimatoprost Concentration in Eluting Drops from a Bottle Packed with 0.1g of 75:25 HEMA-MAA Particles

A gel 75:25 HEMA-MAA was prepared using 0.35 mL of HEMA monomer, 1.05 mLof MAA monomer, 1 mL of a cross linker (SR9035), 12 mL of deionized (DI)water, and 20 μl of photo initiator Darocur® 1173 that were mixed in a20-ml vial and put under UV light and constant stirring to produceparticles. A filter tip was prepared by first inserting in a layer of 11micron pore size filter paper and 0.06 g of p-HEMA particles. Theparticles were compressed and then covered with filter cloth. The bottlewas then filled with 5 mL of 0.01% Bimatoprost/PBS 1×. Drops was dosedout and measured by UV-vis spectrophotometry and the percent uptake wasdetermined relative to that of a drop that did not pass through afilter. As illustrated in FIG. 31, after dispensing of 10 drops, littleor no additional Bimatoprost absorbed in the gel particles.

Bimatoprost Concentration in Eluting Drops from a Bottle Packed with 0.1g of 75:25 HEMA-MAA Particles Loaded with 300 ppm BAK

A gel 75:25 HEMA-MAA was prepared using 0.35 mL of HEMA monomer, 1.05 mLof MAA monomer, 1 mL of a cross linker (SR9035), 12 mL of deionized (DI)water, and 20 μl of photo initiator Darocur® 1173 that were mixed in a20-ml vial and put under UV light and constant stirring to produceparticles. A 1 g portion of the particles were placed into 3 g of1×BAK/water solution. Full uptake of the BAK after 10 days yielded aconcentration of 3× on the particles. A filter tip was prepared by firstinserting in a layer of 11 micron pore size filter paper and 0.06 g ofp-HEMA particles. The particles were compressed and then covered withfilter cloth. The bottle was then filled with 5 mL of 0.01%Bimatoprost/PBS 1×. Drops was dosed out and measured by UV-visspectrophotometry and the percent uptake was determined relative to thatof a drop that did not pass through a filter. As illustrated in FIG. 32,after dispensing of 8 drops, most Bimatoprost passed the gel particles.

Partition Coefficient of Bimatoprost in 25:75 HEMA-MAA Gel Particles

A partition coefficient for Bimatoprost in 25/75 pHEMA/tBM gels foundthat the gels had a very low partition coefficient (K) for bimatoprostof 0.2±0.1 and a partition coefficient of 0.5±0.2 with 3×BAK.

Bimatoprost Concentrations in Eluting Drops from a Bottle Packed with0.1 g of 75:25 tBM-MAA Gel Particles

A gel was prepared from 1.5 mL tBM and 0.5 mL MAA, with 10 μL ofdiethylene glycol dimethacrylate and 6 mg of Darocur TPO upon curing in50 micron thick molds under UV light. The polymerized mixture waspulverized into a fine powder. A filter tip was prepared by inserting ina layer of 11 micron pore size filter paper and placing 0.06 g of p-HEMAparticles into filter tip. These particles were compressed and thencovered with filter cloth. A bottle was then filled with 5 mL of 0.01%Bimatoprost/PBS 1× and drops were dosed and measured using UV-visspectrophotometry with comparison to a drop that was not passed throughthe filter. As shown in FIG. 32, only small amounts of Bimatoprost wereabsorbed in the gel particles.

BAK Removal from Commercial Eye-Drop Formulations

An eye drop bottle's plug (tip) was packed with 0.1 g of p-HEMAparticles for Timolol Maleate commercial formulation (Sandoz Inc.) and0.1 g of p-HEMA/MAA particles for Bimatoprost commercial formation(Allegran Inc.). Approximately 0.5 mL of commercial formulation wasdosed from the eye drop bottle for each measurement with 0.5 mL of afiltered formulation withdrawn by a standard 3 mL syringe for pendantdrop measurements. A drop shape analysis was conducted by theTensiometer to extract surface tension data of the filtered formulation.A calibration curve with equilibrium interfacial surface tension data asa function of BAK concentration was used to estimate concentrations andfractional BAK removal from the filtered eye drop formulation. Periodicsurface tension measurements of the formulations were done to monitorfractional BAK removal. FIG. 33 shows the interfacial surface tension ofthat fits a Langmuir surfactant adsorption isotherm model that allowsestimation of BAK concentrations by the surface tension.

The steady state Langmuir adsorption isotherm model and Langmuir surfaceequation of state were used to fit the equilibrium interfacial surfacetension data are given below:

$\Gamma_{eq} = \frac{\left\lbrack {\left( \frac{\beta}{\alpha} \right)\Gamma_{\infty}c} \right\rbrack}{\left\lbrack {{\left( \frac{\beta}{\alpha} \right)\Gamma_{\infty}c} + 1} \right\rbrack}$${\gamma_{0} - \gamma} = {\left. {{- {RT}}\; \Gamma_{\infty}{\ln \left( {1 - \frac{\Gamma_{eq}}{\Gamma_{\infty}}} \right)}}\rightarrow{\gamma_{0} - \gamma} \right. = {{RT}\; \Gamma_{\infty}{\ln \left( {1 + {\left( \frac{\beta}{\alpha} \right)c}} \right)}}}$

Where a least square error minimization protocol was used to fit theexperimental equilibrium surface tension values and the calculatedestimates using the above model. The fit parameters Γ_(∞) (maximumsurface coverage) and β/α (ratio of kinetic rate constants) wereestimated to be 0.003309 mol/m² and 462.14 m³/mol respectively.Benzalkonium Chloride Removal from Commercial Bimatoprost Formulation(Allegran Inc.)

A particulate gel comprising 25 v/v % HEMA and 75 v/v % MAA was preparedand tested for the removal of BAK using a commercial Bimatoprostformulation having 0.1 mg/mL Bimatoprost and 0.2 mg/mL BAK in a pH 7±0.5sodium phosphate buffer. FIG. 34 shows the interfacial surface tensionmeasured for 15 drops of 33.33 μL and FIG. 35 the % BAK removed,measured using UV-vis spectrophotometry, from the solution on passingthrough a tip loaded with the pulverized particulate gel. Thepolymerized mixture was pulverized into a fine powder. Again high levelsof BAK removal were observed.

Pre-Loading the Filter with BAK or an Alternative Preservative

Based on the US Code of Federal Regulations, Title 21, Volume 4(21CFR200.50), section 200.50 on ophthalmic preparations and dispensers,“all preparations offered or intended for ophthalmic use, includingpreparations for cleansing the eyes, should be sterile.” It is furtherevident that such preparations purport to be of such purity and qualityas to be suitable for safe use in the eye”

As the applied pressure on the eye drop bottle is removed afterinstilling an eye drop, the remaining liquid at the tip is drawn backinto the bottle. This liquid drop could carry bacteria with it. In anormal eye drop bottle, the bacteria would enter the solution where theBAK would keep the solution preserved, preventing bacterial growth. Inthe bottle with the plug, the bacteria may get trapped in the plug whereit could potentially grow. To avoid this possibility, the plug must be asterile environment. To achieve a sterile environment, BAK wasincorporated into the plug by soaking the material comprising the pluginto BAK solutions prior to assembling the plug or by eluting a certainvolume of the BAK solution through the plug after assembly. Although BAKis a preservative, surprisingly a pHEMA plug loaded with BAK provides asterile environment even though the BAK is adsorbed into the polymermatrix and not in the void space in the plug.

The effect of BAK preloading in the pHEMA particles was examined todetermine the maintenance of sterility. BAK was preloaded into pHEMAparticles prepared by heat-initiated polymerization, and the plug ofthese particles integrated in the eye drop prototype was filled withabout 10⁷ cfu/mL Escherichia coli (E. coli, a strain of XL1-Blueobtained from Stratagene, Santa Clara, Calif.) in PBS. The plug wasincubated at 37° C. for 24 hours to see if E. coli survived, flourished,or diminished under the BAK preloaded environment. Preloading theparticles with BAK was carried out by soaking about 80 mg of pHEMAparticles in 166 μg/mL of BAK/PBS solution for 7 days. Based on thepartition coefficient of BAK in pHEMA particles prepared byheat-initiated polymerization being about 200-250, and the density ofthe particles being about 1.2 g/mL, the particles load to about 1 mg ofBAK, i.e., a concentration of about 1.25%, compared to 0.004-0.0025% inmost formulations. The high partition coefficient allows significant BAKuptake into the material without any risk of toxicity from elution ofBAK into the eyes. Alternately, this concentration is achieved bypassing 8 mL (half a typical eye drop bottle volume) of 0.12 mg/mL ofBAK solution through the pHEMA plug. The BAK preloaded particles werethen packed in the prototype bottle with a packed length of 8 mm as thedevice shown in FIG. 19. The eye drop bottle was filled with PBSsolution contained with 10⁷ cfu/mL of E. coli. After three drops of theE. coli contained solution were squeezed through the packed particles,the tip including the packed particles was detached from the bottle suchthat the solution was retained in the plug. The tip was incubated at 37°C. for 24 hours. After 24 hours of incubation, the tip was attached toanother clean eye drop bottle contained with fresh PBS solution. Threedrops of fresh PBS solution was pushed through the packed particles towash out the solution residing in the plug. The three drops createdbefore and after incubation were both collected and properly diluted ifneeded to determine the concentrations of E. coli within the drops. Theconcentrations were determined by drop plating on agar and counting thecolonies on the agar plates. As control, the same experiment procedurewas repeated for pure pHEMA particles without preloading BAK.

Table 6, below, summarized the sterile test results. The initialconcentration of E. coli in the solution is about 10⁷ cfu/mL. To ensurethat E. coli did not get trapped in the filter plug, the concentrationof E. coli in the three drops squeezed through the pHEMA plug weremeasured. The concentration of E. coli after passing through the plugwas in the same order as initial concentration, which indicated that thepore size of few microns could not trapped the bacteria. The solutionremaining in the plug was incubated for 24 hours and the plug washedwith three drops of fresh PBS. The solution washed from the plug wascollected and its concentration of E. coli was determined. As shown inTable 6, without preloaded with BAK, the washed solution has a high E.coli concentration of 13.30×10⁶ cfu/mL, although this concentration doesnot represent the actual concentration of E. coli remaining in the plug.The empty space in the plug was about 20 μL, but one single drop offresh PBS is about 30 μL, such that the 3 drops of fresh PBS leads to asignificant dilution and the actual concentration of the solutionremaining in the plug could be 4 to 5 times higher. This resultindicates that pHEMA particles that are not preloaded with BAK, allowsgrowth of microorganism in the plug. On the other hand, if the particleswere preloaded with sufficient BAK, most of the E. coli does not survivein the filter plug, and the concentration became undetectable. USFederal Regulations require that ophthalmic preservatives achieve 1.0and 3.0 log reduction by days 7 and 14, respectively, along with noincrease in survivors from days 14-28 and no increase in survivors forthe fungi from day 0 to day 28 after inoculation with 10⁶ colony formingunits (cfu)/mL. The plug loaded with BAK performed significantly betterthan the regulatory requirements suggesting that the sterility could beachieved at a lower starting concentration of BAK in the plug. With eachinstillation of eye drop, the concentration of BAK in the plug increaseswhich will improve the degree of sterility.

TABLE 6 Concentration of E. coli as determined by colony countConcentration Concentration Initial concentration after in the plugbefore passing passing through after incubation through the the plug for24 hours Plug material plug (10⁶ cfu/mL) (10⁶ cfu/mL) (10⁶ cfu/mL) PurepHEMA 9.83 9.93 13.30 particles pHEMA particles 9.83 15.40 0 preloadedwith BAK^(a) ^(a)The BAK loading concentration in particles is about12.35 μg/mg = 1.23% (w/w) compared to 0.004-0.025% in the formulations.

In an alternate embodiment of the invention, one can load the filterplug with an additional preservative. The second preservative will bechosen to be: ocularly compatible; of a larger molecular weight thanBAK; and have a lower affinity for the filter material compared to BAK.When the filter is loaded with this preservative, the larger molecularweight will prevent it from diffusing out during the eye dropinstillation. However it will slowly diffuse out possibly in very smallquantities into the liquid remaining in the filter after the eye drop isinstilled to render it sterile. The small amount of the preservativethat diffuses out will eventually be instilled into the eye in the nextcycle of eye drop administration but this amount can be minimized byminimizing the volume of the tip filter. The volume for the filter is10-300 microliter.

The sterile plug can be used for other purposes in addition topreservative removal. It could for example be useful for minimizingoxygen entry into the container when including oxygen scavengingmaterials. This can protect easily oxidizable formulations. The oxygenscavenging material can be integrated into the plug by incorporatingparticles that scavenge oxygen along with the sterile particlescomprising the plug or the oxygen scavenger can be a separate layerabove or below the sterility imparting and/or BAK sequestering material.Oxygen scavenging materials can include iron or ferrous carbonatecombined with sodium chloride or other metal halide, ascorbate, sodiumhydrogen carbonate, or other scavengers, which can be within the plugmaterial or included in another polymeric matrix. The sterile plug canbe used to maintain sterility of the formulation without including anypreservative in the formulation. Any contaminates that enter through theplug get trapped in the pores of the plug and get killed by thepreservative loaded in the plug. To further ensure that themicroorganisms that enter the plug are retained, the plug can bedesigned to prevent drainage of the fluid back into the container byincluding values or alternatively by choosing the pore size such thatthe Young Laplace pressure across the meniscus supports the vacuum inthe container, essentially creating a surface-tension seal.Alternatively, rough particles could pin the contact line, trappingliquid in the plug. Employing materials with a variety of pore sizes canpermit liquid drainage that occurs quickly from the largest pore tocreate an air channels that will equalize the pressure, preventing anyfurther drainage. As an example, a plug packed with particles remainsfully filled with water even after the pressure on the eye drop bottlehas been released. Retaining the fluid in the plug in the interimbetween successive instillations can sequester preservatives or othercomponents that adsorb slowly on the polymer. When the plug remainsfilled with fluid at all times, drops squeezed from the device havecontacted the plug material for periods of a few hours to a day,compared to a few seconds when the plug dried in the interim periodbecause of drainage back to the container.

Incorporation of One-Way Valves in the Bottle.

If a bottle has the plug contacting the liquid, there will be a slowuptake of preservative. Typically, months are required for the BAK toabsorb into the filter because of long diffusion lengths. A valve canalso be placed in the bottle immediately preceding the filter plug toallow flow only when a critical pressure is exceeded. A valve may beincorporated in the side of the bottle to allow air to be included whenthe pressure for drop dispensing is removed. This valve allows pressureequilibration through flow in of air rather than draining back of thefluid.

BAK Dilution in the Bottle

As the applied pressure on the eye drop bottle is removed afterinstilling an eye drop, the remaining liquid in the plug can be suckedback in due to the vacuum created in the bottle. This liquid is devoidof BAK and so its drainage back into the bottle will dilute the BAKconcentration. This effect becomes particularly significant towards theend when only a few drops are left in the bottle. This dilution effectcan be minimized by using plugs with very small void volume. Plugs withvolumes less than three times of that of the eye drops and mostpreferably less than one eye drop are advantageous. To avoid BAK freesolution from draining back into the bottle, either using a valve or bycreating hydrophobic channels in the plug so that water cannot be drawnthrough but air can, alleviating the driving force for fluid to drainback. The higher the BAK concentration that is used the less one needsto compensate for dilution by solution drawn back into the bottle.Finally, if all these design features are not sufficient to preventsignificant dilution, an additional embodiment of the design is to placea preservative loaded membrane in the eye drop bottle so that themembrane can serve as a reservoir to keep the preservative concentrationrelatively unchanged. The membrane could be made from HEMA andpre-equilibrated with BAK at the same concentration as the formulationin the bottle. The preferred location for the membrane is at the bottomof the container to permit full contact with the formulation, but othershapes could be used, e.g. a large particle added to the formulation.The preferred volume of the film will be about 1-5% of the startingvolume of the eye drop formulation. Based on a partition coefficient of300, a 1-5% volume fraction will imply that the film contains 3-15 timesthe amount of BAK in the formulation, thereby proving a strong bufferingeffect protecting against any possibility of dilution of thepreservative.

The ability to equilibrate various composition HEMA/MMA particles with adrug for a period of time to saturate preservative removing particles isillustrated by the drop in the percent drug uptake per drop in: FIGS.36-38 for non-equilibrated, two-week equilibrated, and five-dayequilibrated particles with timolol maleate solution, respectively;FIGS. 39-40 for non-equilibrated and one-week equilibrated particleswith Visine solutions, respectively; FIGS. 41-42 for non-equilibratedand one-month equilibrated particles with Visine A solutions,respectively; and FIGS. 43-45 for non-equilibrated and various-dayequilibrated particles of various HEMA/MMA compositions with Bimatoprostsolutions.

A SOP for Drop Measurement is Below.

Single Eye Drop Measurement and Analysis by UV-Vis Spectrophotometry

1. Purpose

This Standard Operating Procedure (SOP) describes the equipment andprocess used to analyze the concentration of a reagent (or multiplereagents) in a single drop released from an eye drop bottle. This SOP isapplicable to eye drop bottles with or without filters. Thequantification of the concentration in the eye drop allows for thecalculation of percent uptake of the reagent by an inserted filter.

2. Materials

-   -   2.1. Eye drop bottle    -   2.2. Eye drop bottle tip (with or without filter)    -   2.3. Eye drop bottle cap    -   2.4. 0.5-5 mL Pipette (Fisherbrand Elite)    -   2.5. 100-1000 μL Pipette (Fisherbrand Elite)    -   2.6. 20-200 μL Pipette (Fisherbrand Elite)    -   2.7. Pipette tips (Fisherbrand Elite)    -   2.8. Micro Quartz Cuvette, White Wall, 0.4 mL, 10 mm, Cell,        Cuvettes, Spectrometer, 1 cm (Science Outlet)    -   2.9. Mass balance (Denver Instrument M-220D)    -   2.10. UV-Vis Spectrophotometer (ThermoSpectronic Genesys 10 UV)

3. Reagents

-   -   3.1. Phosphate Buffered Saline, 1×[PBS] (Corning)    -   3.2. Ethanol (200 proof, Fisher Scientific)    -   3.3. De-Ionized Water [DI Water]    -   3.4. Eye drop bottle formulation (varies per experiment)

4. Procedure

-   -   4.1. Cleaning Cuvette    -   This section's steps will be repeated throughout the procedure.        When used, this section will be referenced        -   4.1.1. Remove any residual liquid inside of cuvette        -   4.1.2. Fill cuvette with DI water, then empty cuvette        -   4.1.3. Fill cuvette second time with DI water, then empty            cuvette        -   4.1.4. Fill cuvette with ethanol, then empty cuvette        -   4.1.5. Fill cuvette with DI water, then empty cuvette        -   4.1.6. Fill cuvette with DI water, then empty cuvette        -   4.1.7. Air dry cuvette until dry    -   4.2. Eye drop bottle assembly        -   4.2.1. If not previously assembled, gather eye drop bottle,            tip, and formulation        -   4.2.2. Insert formulation into eye drop bottle        -   4.2.3. Insert tip into bottle        -   4.2.4. Cap bottle        -   4.2.5. If equilibration is required, proceed to section 4.3.            If not, skip to 4.4    -   4.3. Equilibration (filter only)    -   Equilibration allows for contact time between the formulation        and the filter to saturate the filter with the desired reagent        to prevent uptake during eye drop use.        -   4.3.1. Carefully invert bottles so that cap and tip are            pointed downward        -   4.3.2. Mark start time and keep inverted for desired period            of time        -   4.3.3. Periodically examine bottles to ensure no leakage of            formulation        -   4.3.4. After desired timespan, return eye drop bottles to            upright position        -   4.3.5. Proceed to section 4.4 to measure eye drop    -   4.4. Eye Drop Measurement        -   4.4.1. Clean outside of cuvette with Di water        -   4.4.2. Follow section 4.1 for cleaning interior of cuvette        -   4.4.3. Fill cuvette with PBS        -   4.4.4. Insert cuvette into UV-vis spectrophotometer        -   4.4.5. Close UV-vis spectrophotometer and set blank            -   Note: Wavelength and UV-vis settings will depend upon                formulation used        -   4.4.6. After blank is set, remove cuvette        -   4.4.7. Follow section 4.1 for cleaning procedure        -   4.4.8. Place clean cuvette on mass balance        -   4.4.9. Tare        -   4.4.10. Take eye drop bottle, invert, and hold over cuvette        -   4.4.11. Gently squeeze eye drop bottle until a single drop            falls into the cuvette        -   4.4.12. Record mass of drop in cuvette        -   4.4.13. Return eye drop bottle to storage        -   4.4.14. Calculate required mass of PBS for dilution            -   Note: This amount of added PBS will vary based on                desired dilution        -   4.4.15. Add required mass of PBS into cuvette using            appropriate pipet and pipet tip, record added mass        -   4.4.16. Gently shake cuvette to mix        -   4.4.17. Place cuvette inside of UV-vis spectrophotometer        -   4.4.18. Close UV-vis and measure sample        -   4.4.19. Record data        -   4.4.20. Remove cuvette and clean following section 4.1.        -   4.4.21. If measuring second sample with same formulation,            start at step 4.4.8

5. Data Analysis

This procedure collects the spectra of a diluted drop of formulationsolution after exiting an eye drop bottle. In order to calculate theconcentration, the spectra must be compared to a calibration curve,which is the measured spectrum of a known concentration solution. Thetwo are compared to find the ratio between the spectra height of themeasured curve and the calibration curve, which is the same ratio astheir concentrations. For this procedure, the calibration curve wasgathered by the procedure laid out in section 4.4, but on a solution ofknown concentration, usually the starting solution. This solution wasnot sent through any filter and showed the case of no uptake ofsolution.

Once a drop has been measured and compared with the calibration curve,it can be converted into concentration, which, when accounting fordilution, can show the amount of drug taken up. This fraction ofdisappeared mass is considered the percent uptake by the filter.

Standard Operating Procedure for BAK Removal.

Purpose/Background

The purpose of this procedure is to provide information for evaluationof Benzalkonium chloride removal from commercial eye drop formulations.Commercial multi-dose ophthalmic formulations have an added preservativecontent, namely Benzalkonium chloride to maintain sterility of theformulation. A high frequency of administration of multi-doseformulation leads to an increase in systemic uptake of suchpreservatives. This causes irreversible damage to the cornea. A filtermade from p-HEMA or p-HEMA/MAA particles is designed for delivering safemulti-dose preservative-free formulations. Since the concentration ofBAK is significantly low in the filtered formulation, interfacialsurface tension data is used to evaluate the fractional removal ofpreservative from the formulation. The procedure requires only a minimalamount of background in pendant drop tensiometer to follow theprotocols, while at the same time a sufficiently complete description toperform detailed surface tension measurements necessary for evaluationof BAK removal.

Chemicals:

-   -   Monomer: 2-hydroxyethyl methacrylate (HEMA, 97%) monomer and        Methacrylic acid (99%) from Sigma-Aldrich Chemicals (St. Louis,        Mo., USA)    -   Crosslinker: ethoxylated (15) trimethylolpropane triacrylate        (SR9035) obtained from Sartomer (Warrington, Pa., USA)    -   Photo-initiator: Photo-initiators Darocur® 1173 by Ciba        Specialty Chemicals (Tarrytown, N.Y., USA)    -   Ethanol (200 proof) from Decon Laboratories Inc. (King of        Prussia, Pa., USA)    -   Benzalkonium chloride from MP Biomedicals, LLC    -   De-ionized water

Materials and Equipment:

-   -   Whatman© International limited filter paper size 1 (11 cm        diameter, 11 μm pore size)    -   Luer Lok tip syringes from BD, Franklin Lakes, N.J., USA    -   14-gauge, 1.5″ precision applicator dispenser needle from        Creative Hobbies    -   Standard 30 ml eye-drop bottle from Topwell Inc., Lexington,        Ky., USA

Procedures

Preparation of the Filter Bed

-   -   Detach the standard tip or plug of the designed eye drop bottle.    -   Check the plug (dropper tip) to make sure that it is not chipped        or cracked.    -   Fill the plug's nozzle with two layers of filter paper. Make        sure that the filter paper (pre-cut based on nominal diameter of        the plug's nozzle) covers the nozzle. This is to ensure that        finer particles from the packed filter are not dispensed along        with the filtered eye-drop formulation.    -   Measure approximately 0.1 g of pre-made p-HEMA or p-HEMA/MAA        particles. Pack the area beneath the plug's nozzle with the        particles.    -   Cover the base of the plug with a layer of filter cloth to        ensure that they stay intact within the plug.    -   Gently tap the layer of filter cloth with a tweezer to ensure        that it stays intact near the plug's base.    -   Mount the filter/packed plug on to the eye-drop bottle's neck to        complete the proposed design.    -   Make sure all eye-drop bottles are labelled with contents and        the type of packed particles.

General Guidelines for Cleaning Particles

-   -   Detach the plug packed with particles from the designed eye drop        bottle.    -   Transfer 10-15 ml of Dulbecco's phosphate buffered saline (PBS)        into the eye drop bottle and mount the packed plug back on to        the bottle.    -   Gently squeeze the eye-drop bottle to withdraw 10 ml of the        transferred phosphate buffered saline from the eye-drop bottle.        This step ensures that impurities in the filter bed gets leached        out upon exposure to PBS.    -   Remove the cleaned filter from the designed eye drop bottle.    -   Rinse the eye-drop bottle with DI water and air-dry it prior        transferring the eye-drop formulation.    -   Transfer 10-15 ml of commercial eye-drop formulation (0.01%        Benzalkonium chloride) into the eye drop bottle and mount the        cleaned filter back on to the bottle.

Guidelines Prior Surface Tension Measurements

-   -   Gently squeeze the eye-drop bottle with an embedded filter to        withdraw 0.5 ml of the commercial eye-drop formulation. An        initial dose of 0.5 ml is withdrawn to avoid dilution of the        filtered formulation.    -   Dose out a volume of 0.5 mL (approximately 15 drops of 33 μl        each) for measuring the surface tension of the filtered        formulation. After a period of 24 hours, withdraw another batch        of filtered formulation (0.5 mL) and monitor the surface tension        of filtered formulation.    -   Standard 5 ml vial or a microplate is used for collecting the        filtered formulation for surface tension measurements. Rinse the        vial or the surface of the microplate with acetone and DI water        prior collecting the dosed formulation.    -   Using a new Luer lock syringe and a needle, withdraw the dosed        formulation from the vial or microplate.

Surface Tension Measurements Using Pendant Drop Tensiometer

This section is intended for users of DSA Kruss Pendant drop tensiometerand DSA v 1.9 Drop Shape Analyzer, helping them perform interfacialsurface tension measurements.

-   -   Switch on the DSA100 Pendant drop tensiometer. At the time of        writing, DSA v. 1.9 is the software package used to operate the        tensiometer for surface tension measurements. Start the DSA1        software with shortcut symbol. The following illustration shows        the user interface of the DSA software.    -   Ensure that the angle of inclination of the tilt is set to 0°    -   Select the following menu item FG>Acquire to set image        transmission to live mode. Alternatively, the shortcut key F5        can also be used to do the same.    -   In the menu under Options, select in sequence the options Drop        Type and Sub Type. Make sure that the drop type is selected as        Pendant Drop [PD] and for subtype; the configuration of the drop        is set as Top->Bottom.    -   Fit the Luer lock syringe containing the dosed formulation in        the manual deposition system. The following illustration shows a        pre-filled syringe positioned in the deposition system. If the        tip of the syringe's plunger is misaligned with the deposition        system, click on Refill tab under the DSA device control panel.        This moves the position of the knob present in the deposition        system upwards to allow space for the plunger.    -   Move the position of the needle downwards until it appears in        the image. This can be done by adjusting the position of the        scroll bar present in the device control panel. Alternatively,        the position of the needle can also be controlled by the        shortcut keys Page Up and Page Down.    -   Regulate lens zoom so that the image of the needle occupies the        center of the frame. This is done by rotating the “Zoom” knob at        the top left of the DSA 100 equipment. To adjust the sharpness        of the image, click on Options>Focusing Assistant and tune the        focus knob at the top left of the DSA100 equipment. The field        “Median” is color coded and should appear in green, indicating a        large numerical value. A good range for the median value is        75-80. Alternatively, the focal length can be adjusted by using        the shortcut keys Home and End respectively.    -   Select the Dosing tab in the device control panel. The        formulation in the syringe can be dosed out using the two action        buttons present in the dosing tab. The direction of the arrows        corresponds to the movement of the syringe plunger. Click on the        button marked with up arrow to dose out a drop from the syringe.    -   Make sure that the dosing mode is set to continuous. The dosing        speed can be entered by using the input field or the sliding        controller. Since the volume of the filtered formulation in the        syringe is only 0.5 ml, the recommended flow rates to be set is        from 20-200 μl/min. A higher dosing speed is not suitable for        drop production but only for emptying the contents in the        syringe.    -   Adjust the zoom and needle height so that the drop occupies as        much as 80% of the whole frame height. The image contains three        colored lines. These lines define the region of drop curvature        that the software uses to evaluate the surface tension of the        formulation. They can be moved by keeping the mouse key pressed        down.    -   Make sure that the top two lines are positioned within the        region of the needle. The width of the needle is measured        between these two lines. The lower line is placed slightly below        the transition point between the formulation drop and the        needle. The software uses the drop curvature below this line for        evaluation of surface tension.    -   Manual Calibration based on needle width: A standard image of        the drop contains 768 pixels with respect to a horizontal width        of 8 inches of the image. The nominal outer diameter of the        14-gauge 1.5″ precision needle used for the pendant drop        measurements is 0.5144 mm. A custom software can be used to        import the drop image and estimate the needle's width in inches.        The scale of the image or magnification factor is calculated        based on the needle diameter.

${{Magnification}\mspace{14mu} {{factor}\mspace{14mu}\lbrack{MAG}\rbrack}} = {{\frac{\begin{matrix}{{Number}\mspace{14mu} {of}\mspace{14mu} {pixels}\mspace{14mu} {contained}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {image}\mspace{14mu} {with}} \\{{respect}\mspace{14mu} {to}\mspace{14mu} {horizontal}\mspace{14mu} {{width}\mspace{14mu}\lbrack 768\rbrack}}\end{matrix}}{{Horizontal}\mspace{14mu} {width}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {drop}\mspace{14mu} {{image}\mspace{14mu}\left\lbrack 8^{''} \right\rbrack}} \times \frac{\begin{matrix}{{Needle}\mspace{14mu} {width}\mspace{14mu} {based}\mspace{14mu} {on}\mspace{14mu} {the}\mspace{14mu} {region}\mspace{14mu} {it}} \\{{occupies}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {drop}\mspace{14mu} {{image}\mspace{14mu}\left\lbrack x^{''} \right\rbrack}}\end{matrix}}{{Nominal}\mspace{14mu} {outer}\mspace{14mu} {diamter}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} 14\text{-}{gauge}\mspace{14mu} {{needle}\mspace{14mu}\left\lbrack {0.5144\mspace{14mu} {mm}} \right\rbrack}}} = {Y\mspace{14mu} {pixels}\text{/}{mm}}}$

-   -   In the menu under Options, click on Drop Info and check the        values of parameters under their respective input fields. Make        sure that the needle diameter and the calculated magnification        factor are set to the right values. If the surface tension of        the formulation is measured based on fluid-air interface, the        density of the embedding phase is set to the density of air.    -   Once all the parameters are set, click on the symbol present in        the symbol bar beneath the menu. DSA1 determines and extracts        the drop shape which is indicated by a green/red contour        surrounding the drop.    -   Click on the symbol in the symbol bar. The surface tension of        the formulation is calculated using a Young-Laplace fit. The        measured value appears in the Results window.    -   To monitor the surface tension of the formulation as a function        time i.e., measure the dynamic surface tension of the        formulation, click on Tracker-man under options. Enter the        duration of the dynamic measurement and make sure that the        following item “Extract Profile and Calculation” is checked.        Start the feature to obtain the estimates of interfacial surface        tension of the formulation at regular time intervals.        -   After a period of 24 hours, withdraw another batch of            filtered formulation, 0.5 ml (approximately 15 drops of 33            μl each) and monitor the surface tension of filtered            formulation. Repeat the measurements till 10 ml of the            formulation is dosed out.            Below is a SOP for the preparation of hydrogel particles.

Preparation of Hydrogel Particles for Eye Drop Filters 1.0 Purpose

-   -   This SOP describes the production with multiple ratios of        methacrylic acid (MAA) and 2-hydroxyethylmethacrylate (HEMA) to        make particles for the uptake of benzalkonium chloride (BAK) in        filter tips designed for ophthalmic drug solutions.

2.0 Reagents and Materials

2.1 Chemicals

-   -   2.1.1 Monomer: 2-hydroxyethyl methacrylate (HEMA, 97%) monomer        and Methacrylic acid (99%) from Sigma-Aldrich Chemicals (St.        Louis, Mo., USA).    -   2.1.2 Crosslinker: ethylated (15) trimethylolpropane triacrylate        (SR9035) obtained from Sartomer (Warrington, Pa., USA)    -   2.1.3 Photo-initiator: Photo-initiators Darocur® 1173 by Ciba        Specialty Chemicals (Tarrytown, N.Y., USA).    -   2.1.4 Ethanol (200 proof) from Decon Laboratories Inc. (King of        Prussia, Pa., USA).    -   2.1.5 De-ionized water.

2.2 Materials and Equipment:

-   -   2.2.1 liter beaker (Fisher Industries)    -   2.2.2 Magnetic stirrer (approximately 5 cm*0.6 cm)    -   2.2.3 Spatula    -   2.2.4 Para-film (Bemis laboratory film 4′*4′)    -   2.2.5 Mortar (13 cm*5 cm) and pestle (13 cm*3 cm)    -   2.2.6 Whatman© International limited filter paper size 1 (11 cm        diameter, 11 μm pore size)    -   2.2.7 55×17 mm (diameter×height) Pyrex® petri dish.    -   2.2.8 UVB-10 transilluminator (ULTRA•LUM INC, Carson, Calif.,        USA) with an intensity of 16.50 mW/cm² sharply peaked at 310 nm.    -   2.2.9 Welch 2546B-01 Standard duty vacuum filter.    -   2.2.10 Nalgene© 180 PVC non-toxic autoclavable LAB/FDA/USP VI        grade (⅜″ ID).    -   2.2.11 Corning Pyrex® 125 ml micro-filter conical flask.    -   2.2.12 Coors Coorstek© 320 ml 90 mm ceramic porcelain Buchner        vacuum filter funnel.

3.0 Procedure

3.1 Particle Preparation Steps (50 g Batch Size)

(Monomer—100% HEMA)

-   -   3.1.1 Mix 42 ml (1 T) of HEMA monomer, 3 ml (0.07 T) of        crosslinker SR9035, 360 ml (8.5 T) of deionized (DI) water in a        1 liter beaker.    -   3.1.2 Stir the mixture using a magnetic stirrer for 20 minutes        at 900 rpm at room temperature.    -   3.1.3 Deoxygenate the mixture by bubbling with pure nitrogen for        30 min.    -   3.1.4 After the degassing step, add 300 μl (0.007 T) of        photoinitiator Darocur® 1173.    -   3.1.5 The mixture is then irradiated with UV light for 2 hours        by a UVB-10 transilluminator        -   (ULTRA•LUM INC, Carson, Calif., USA) with an intensity of            16.50 mW/cm² sharply peaked at 310 nm.    -   3.1.6 During the UV curing, make sure that the top of the beaker        is covered with parafilm sheet to avoid water evaporation and        oxygenation. Also, the mixture is continuously stirred using a        magnetic stirring bar at about 90 rpm.    -   3.1.7 After the polymerization step, the mixture is stirred at        about 1500 rpm (using big motor stirrer) to disintegrate the gel        so formed.    -   3.1.8 The gel is then separated from the solution by vacuum        filtration method and washed with a large quantity of DI water.    -   3.1.9 The gel is then left to dry for 24 hrs. at 130-140° F.    -   3.1.10 Crush the gel so obtained using a mortar and pestle to        obtain the particles.

3.2 Particle Preparation Steps (50 g Batch Size)

(Monomers—50% HEMA+50% Methacrylic Acid)

-   -   3.2.1 Mix 42 ml (1 T) of the 2 monomers (21 ml HEMA+21 ml        Methacrylic Acid), 3 ml (0.07 T) of crosslinker SR9035, 360 ml        (8.5 T) of deionized (DI) water in a 1 liter beaker.    -   3.2.2 Stir the mixture using a magnetic stirrer for 20 minutes        at 900 rpm at room temperature.    -   3.2.3 Deoxygenate the mixture by bubbling with pure nitrogen for        30 min.    -   3.2.4 After the degassing step, add 300 μl (0.007 T) of        photoinitiator Darocur® 1173.    -   3.2.5 The mixture is then irradiated with UV light for 2 hours        by a UVB-10 transilluminator        -   (ULTRA•LUM INC, Carson, Calif., USA) with an intensity of            16.50 mW/cm² sharply peaked at 310 nm.    -   3.2.6 During the UV curing, make sure that the top of the beaker        is covered with parafilm sheet to avoid water evaporation and        oxygenation. Also, the mixture is continuously stirred using a        magnetic stirring bar at about 90 rpm.    -   3.2.7 After the polymerization step, the mixture is stirred at        about 1500 rpm (using big motor stirrer) to disintegrate the gel        so formed.    -   3.2.8 The gel is then separated from the solution by vacuum        filtration method and washed with a large quantity of DI water.    -   3.2.9 The gel is then left to dry for 24 hrs. at 130-140° F.    -   3.2.10 Crush the gel so obtained using a mortar and pestle to        obtain the particles.

3.3 Particle Preparation Steps (50 g Batch Size)

(Monomers—75% Methacrylic Acid+25% HEMA)

-   -   3.3.1 Mix 42 ml (1 T) of the 2 monomers (31.5 ml Methacrylic        Acid+10.5 ml HEMA), 3 ml (0.07 T) of crosslinker SR9035, 360 ml        (8.5 T) of deionized (DI) water in a 1 liter beaker.    -   3.3.2 Stir the mixture using a magnetic stirrer for 20 minutes        at 900 rpm at room temperature.    -   3.3.3 Deoxygenate the mixture by bubbling with pure nitrogen for        30 min.    -   3.3.4 After the degassing step, add 300 μl (0.007 T) of        photoinitiator Darocur® 1173.    -   3.3.5 The mixture is then irradiated with UV light for 2 hours        by a UVB-10 transilluminator (ULTRA•LUM INC, Carson, Calif.,        USA) with an intensity of 16.50 mW/cm² sharply peaked at 310 nm.    -   3.3.6 During the UV curing, make sure that the top of the beaker        is covered with parafilm sheet to avoid water evaporation and        oxygenation. Also, the mixture is continuously stirred using a        magnetic stirring bar at about 90 rpm.    -   3.3.7 After the polymerization step, the mixture is stirred at        about 1500 rpm (using big motor stirrer) to disintegrate the gel        so formed.    -   3.3.8 The gel is then separated from the solution by vacuum        filtration method and washed with a large quantity of DI water.    -   3.3.9 The gel is then left to dry for 24 hrs. at 130-140° F.    -   3.3.10 Crush the gel so obtained using a mortar and pestle to        obtain the particles.

3.4 Particle Cleaning Steps

(Common for all)

-   -   3.4.1 To remove the unreacted monomer part and other impurities,        soak the freshly crushed particles in 800 ml (19 T) of ethanol        for 2 days while stirring the mixture at 300 rpm using a        magnetic stirrer. Make sure to change the solvent every day.        Separate the particles from ethanol using vacuum filtration and        dry them for 24 hrs. at 130-140° F.    -   3.4.2 After ethanol washing, soak the particles in 800 ml (19 T)        of DI water for 4 days (changing water every day) while stirring        the mixture at 300 rpm using a magnetic stirrer. Separate the        particles from water using vacuum filtration and dry them for 24        hrs. at 130-140° F. to obtain the final cleaned particles.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A preservative removing device, comprising a porous hydrophilicpolymeric matrix, wherein the porous hydrophilic polymeric matrixcomprises a material with a hydraulic permeability greater than 0.01 Daand fits an outlet of a container for a solution, emulsion, orsuspension, wherein the solution, emulsion, or suspension is configuredto flow through the porous hydrophilic polymeric matrix, and wherein theporous hydrophilic polymeric matrix is configured to remove at least 50%of a preservative and retain at least 50% of a hydrophobic ophthalmicagent within the solution, emulsion or suspension.
 2. The preservativeremoving device according to claim 1, wherein the porous hydrophilicpolymeric matrix has a partition coefficient for the preservative fromthe solution, emulsion, or suspension of at least
 100. 3. Thepreservative removing device according to claim 1, wherein the poroushydrophilic polymeric matrix comprises particles.
 4. The preservativeremoving device according to claim 1, wherein the porous hydrophilicpolymeric matrix is preloaded with the preservative at 1 to 90% ofsaturation of the preservative in the porous hydrophilic polymericmatrix.
 5. The preservative removing device according to claim 1,wherein the porous hydrophilic polymeric matrix comprises poly hydroxylethyl methacrylate (pHEMA), poly hydroxyl ethylmethacrylate-co-methacrylic acid, or a combination thereof.
 6. Thepreservative removing device according to claim 1, wherein the poroushydrophilic polymeric matrix has interconnected pores, wherein the poreshave an average radius of 1 to 60 μm.
 7. The preservative removingdevice according to claim 1, wherein the porous hydrophilic polymericmatrix is partitioned as microparticles with cross-sections of 2 to 100μm.
 8. The preservative removing device according to claim 1, whereinthe hydraulic permeability is greater than 1 Da.
 9. The preservativeremoving device according to claim 1, wherein the preservative isbenzalkonium chloride (BAK).
 10. The preservative removing deviceaccording to claim 9, wherein the porous hydrophilic polymeric matrix ispreloaded with the BAK at a concentration of one to 100 times that ofthe solution, emulsion, or suspension in the container.
 11. Thepreservative removing device according to claim 9, wherein the poroushydrophilic polymeric matrix 1 is preloaded with a second preservative.12. The preservative removing device according to claim 1, wherein theporous hydrophilic polymeric matrix includes the hydrophobic ophthalmicagent at a level below saturation.
 13. The preservative removing deviceaccording to claim 1, wherein the porous hydrophilic polymeric matrix issaturated with the hydrophobic ophthalmic agent.
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. A multi-dosing device for delivery of anophthalmic solution, comprising: a compressible bottle and apreservative removing device comprising a porous hydrophilic polymericmatrix, a solution comprising a hydrophobic ophthalmic agent, apreservative, and, optionally, a preservative source to maintain thepreservative concentration in the solution; wherein the bottle comprisesan outlet extension, wherein the preservative removing device when dryhas dimensions smaller than the internal dimensions of the outletextension, and wherein the preservative removing device when wet hasdimensions larger than the internal dimensions of the outlet extension.18. (canceled)
 19. (canceled)
 20. (canceled)
 21. The preservativeremoving device according to claim 1, wherein the hydrophobic ophthalmicagent comprises dexamethasone, bimatoprost, or latanoprost.
 22. A methodof administering an ophthalmic agent, comprising: providing acompressible bottle comprising a preservative removing device at theoutlet of the compressible bottle; providing a solution comprising ahydrophobic ophthalmic agent and a preservative; and applying pressureto the compressible bottle, wherein the solution is forced through thepreservative removing device, wherein at least 50 percent of thepreservative is removed from the solution and wherein at least 50percent of the hydrophobic ophthalmic agent is retained by the solution.23. (canceled)
 24. (canceled)
 25. The preservative removing deviceaccording to claim 5, wherein the porous hydrophilic polymeric matrixcomprises poly hydroxyl ethyl methacrylate-co-methacrylic acid andwherein the poly hydroxyl ethyl methacrylate-co-methacrylic acidcomprises 60%-75% methacrylic acid and 25%-40% hydroxyl ethylmethacrylate by volume.
 26. The preservative removing device accordingto claim 5, wherein the porous hydrophilic polymeric matrix comprises across-linker and wherein the cross-linker comprises ethylene glycoldimethacrylate or SR9035.
 27. The preservative removing device accordingto claim 1, wherein the porous hydrophilic polymeric matrix comprisestert-butyl methacrylate and methacrylic acid in a ratio of 25 to 75 byvolume.
 28. The preservative removing device according to claim 1,wherein a partition coefficient of the porous hydrophilic polymericmatrix for the hydrophobic ophthalmic agent is within a range from 10 to50.